JP2003533970A - Regulation of gene expression by neuroleptics - Google Patents

Abstract

  (57) [Summary] Provided are polynucleotides, polypeptides, kits and methods related to genes expressed in the central nervous system that are controlled by a neuroleptic.

Description

Detailed Description of the Invention

This application is a provisional application No. US application filed on October 26, 1999. 60/161
, 379 and US Provisional Application No. 60/186
, 918 claims priority. Both applications are hereby incorporated by reference.

BACKGROUND schizophrenia and dopamine receptors of the invention. Midbrain dopamine neurons have been shown to play an important role in normal and diseased brain function. For example, many psychiatric disorders involve the midbrain striatal dopamine pathway (neurons connecting the striatum and substantia nigra) and the mesolimbic dopamine pathway (ventral tegmental area and often the striatum). Associated with hyperactive dopaminergic activity in the nigrostriatal dopaminergic system associated with limbic systems such as the amygdala, olfactory tubercle and nucleus accumbens, which are thought to be ventral extensions To do. Furthermore, Parkinson's disease is known to be caused by degenerative degeneration of dopamine neurons in the nigrostriatal pathway.

Neuroleptic (antipsychotic) drugs. Neuroleptic drugs such as haloperidol and clozapine are widely used for long-term treatment of various psychoses. The antipsychotic effects of neuroleptics are generally believed to be the result of inhibition of the mesolimbic dopamine D ₂ receptors (Metzler et al., Schizophrenia Bul.
l. 2, 19-76 (1976)). The clearest evidence for this is the excellent correlation observed between the therapeutic effects of neuroleptics and their binding affinity for the D ₂ receptor (Seeman et al., Curr. Opn. Neurol.
And Neurosurg. 6,602-608 (1993); Creese.
et al. , Science, 192, 481-483 (1976); Pe.
routka et al. , Am. J. Psych. 137, 1518-15
22 (1980); Deutsch, et al. , Schizophren. R
es. 4, 121-156 (1991); Seeman, P .; , Synapse
1, 133-152 (1987)). Although neuroleptic drugs have an affinity for other neurotransmitter receptors in the brain, such as muscarinic acetylcholine, 5-HT, alpha-adrenergic and histamine receptors, these receptors are associated with clinical effects. No correlation has been observed (Peroutka et al., Am. J. Ps.
ych. (1980); Richelson et al. , Eur. J. Ph
arm. , 103, 197-204 (1984)).

Studies show that dopamine receptors are blocked by up to 70% only hours after neuroleptic administration (Sedval et al., Arch. Gen. Ps.
ych. 43,995-1006 (1986)). This inhibition has been shown to cause a compensatory increase in the number of dopamine receptors and increased sensitivity to non-inhibitory receptors (
Clow et al. , Psychopharm. 69, 227-233 (1
980); Rupniak et al. , Life Sci. 32,2289
-2311 (1983); Rogue et al. , Eur. J. Pharm
． , 207, 165-169 (1991)). Furthermore, the short-term effects of dopamine antagonists on the brain are well known and such effects include increased dopamine synthesis and catabolism, increased dopamine neuron firing rate due to inhibition of presynaptic dopamine autoreceptors (Grace et al., J. Pharm. Exp. Th
er. , 238, 1092-1100 (1986), and augmented cyclic AMP formation by the postsynaptic dopamine receptor (Rupniak et al.
, Psychopharm. , 84, 519-521 (1984)).

Side effects of neuroleptic drugs. In addition to their antipsychotic effects, neuroleptics can cause a range of mild to severe side effects. Some of these side effects include hypotension and tachycardia due to alpha-adrenergic receptor inhibition, and dry mouth and blurred vision due to muscarinic acetylcholine receptor inhibition.
Due to the extremely disappointing nature of neuroleptics. The major and most undesirable effect associated with neuroleptic treatment is long-lasting movement disorders associated with extrapyramidal side effects (Marsden et al., Psycol. Med. 10, 55-72).
(1980)). Extrapyramidal side effects are associated with inhibition of dopamine receptors in the dorsal striatum (Moore et al., Clin. Neuropharmacol. 1
2, 167-184 (1989)), dystonia (muscle cramps), akathisia (restlessness), Parkinson-like symptoms and movement disorders such as tardive dyskinesia. Approximately 20% of patients taking antipsychotics show Parkinson-like symptoms, and inhibition of striatal dopamine D ₂ receptors is functional for degeneration of nigrostriatal dopamine neurons seen in Parkinson's disease be equivalent to. Tardive dyskinesia is a syndrome of abnormal involuntary movements that suffers from about 25% of patients receiving neuroleptic treatment (Jeste et al., Psychopharmacol. 106).
, 154-160 (1992)). The risk of this side effect is that it is potentially irreversible, i.e. the patient exhibits symptoms of tardive dyskinesia for a long period of time even after quitting the antipsychotic. This represents an epigenetic factor for long-term neuroleptic therapeutic efficacy.

Interestingly, "typical" neuroleptics such as haloperidol and fluphenazine are more susceptible to such side effects than "atypical" neuroleptics, such as clozapine, which rarely cause extrapyramidal side effects. Have a higher tendency to wake up. Although clozapine differs in pharmacological properties from haloperidol, the specific mechanism by which it does not show exercise side effects is unknown. Because clozapine has a high affinity for other neurotransmitter receptors, such as muscarinic, adrenergic and serotonin receptors, the antipsychotic effects of clozapine may be due in part to their inhibition. Yes, that may restore the proper balance of basal ganglia dopamine input and output pathways.

Genetics and genes involved in neuropsychiatric disorders. In the general population, the risk of developing a mental disorder is about 1-2% (Maier, W., and Schwab.
, S. , Molecular Genetics of schizophre
nia. Current Opinion in Psychiatry 1
1: 19-25 (1998); Kendler, K .; S. , Twin stud
ies of psychiatric illness: current s
status and future directions, Arch Gen
Psychiatry 50: 9095-915 (1993)). However, this risk is 10% or 4 if one or both of the parents have the disease, respectively.
Increase to 0%. The consensus in monozygotic and dizygotic twins is only 40-50% (Maier and Schwab (1998)). Although there is no doubt that the transmission of mental disorders has a genetic component, the lack of perfect concordance in dizygotic twins suggests that there are other causative environmental factors. (Maier and Schwa
b (1998); Kendler (1993)). A recent challenge in genetic research for psychosis is the identification of mutations or treatment-related genes that sensitize such disorders. One approach to the latter is to identify genes whose expression changes during drug treatment.

Expression of immediate-early genes by short-term neuroleptic treatment. Despite immediate dopamine receptor occupancy, neuroleptics delay the onset of clinical action, which can often take weeks. Furthermore, as explained above, neuroleptics are characterized by their ability to cause delayed, long-lasting movement disorders. The distinct temporal contradiction that exists between dopamine receptor occupancy and the onset of therapeutic and extrapyramidal side effects suggests that additional molecular changes in the brain occur downstream from dopamine receptor inhibition. In an attempt to identify downstream molecular mechanisms, research has focused on dopamine receptor regulation of individual target genes in the striatum and nucleus accumbens.

For example, some studies have shown that short-term treatment with antipsychotics causes induction of some very early genes (Nguyen et al., Proc.
Natl. Acad. Sci. , 89, 4270-4274 (1992); Ma.
cGibbon et al. , Mol. Brain. Res. 23, 21-3
2 (1994); Robertson et al. , Neuro. Sci. ，
46, 315-328 (1992); Dragunow et al. , Neu
ro. Sci. 37, 287-294 (1990); Miller J. et al. Neu
rochem. , 54, 1453-1455 (1990). Some immediate early gene proteins (IEGPs) act as transcription factors by binding to specific DNA sequences and controlling gene transcription. Therefore, IEGPs can link receptor-mediated signaling effects to long-term genomic activity. Recent studies have shown that a typical neuroleptic, haloperidol, induces c-Fos expression in the rat striatum and nucleus accumbens, whereas the atypical neuroleptic, clozapine, only induces c-Fos in the nucleus accumbens. It was shown to induce (Nguyen et al., Proc. Natl. Acad.
Sci. , (1992); MacGibbon et al. , Mol. Bra
in Res. (1994); Robertson et al. , Neuro
sci. (1992)). Haloperidol is found in the striatum and nucleus accumbens by FosB, J
It has also been shown to induce expression of other IEGPs such as unB, JunD and Krox24 (Rogue et al., Brain Res. Bul.
l. 29, 469-472 (1992); Marsden et al. , Ps
ych. Med. (1980); Moore et al. , Clin. Neu
ropharmacol. (1989)). In contrast, clozapine is K in the nucleus accumbens.
It has been shown to induce nox24 and JunB (Nguyen et al.
al. , (1992); MacGibbon et al. , (1994))
. These results suggest that the lower tendency of clozapine to cause extrapyramidal side effects, as compared to "typical" neuroleptics, may be associated with the inability to induce IEGPs in the striatum.

The emergence of immediate-early genes after short-term treatment with neuroleptic drugs precedes a number of other molecular changes involved in delayed adaptive changes, possibly caused by drug treatment in the striatum.

Changes induced by long-term neuroleptic treatment. Long-term treatment with neuroleptic drugs has also been shown to cause altered expression of certain neuropeptide and neurotransmitter receptors. In distinct regions of the striatum, neurotensin and enkephalin are upregulated by haloperidol long-term (7-28 days) treatment, but protachykinin mRNA levels are reduced (Merchant et al.
al. J. Pharm. Exp. Ther. 271, 460-471 (199
4); Defs et al. J. Neurochem. , 63, 777-
780 (1994); Angulo et al. , Neurosci. Let
t. 113, 217-221 (1990)). In contrast, long-term clozapine treatment causes a decrease in enkephalin mRNA levels and only modest changes in neurotensin and tachykinin expression (Merchant et al., (199).
4); Mercugliano et al. , Neurosci. Lett.
136, 10-15 (1992); Angulo et al. , (1990)
). These differences suggest that neuropeptides may play a role in movement disorders resulting from treatment with typical neuroleptic drugs.

Researchers have also shown regulation of genes associated with glutaminergic neurotransmission. For example, decreased mRNA expression of the glutamate transporter, GLT-1, was observed in the striatum after 30 days of haloperidol treatment, but not in clozapine exposure (Schneider et al., Neurorepor).
t. , 9, 133-136 (1998)). Similar treatment with haloperidol increased the N-methyl-D-aspartate (NMDA) receptor subunits, NR1 and NR2, but lower induction with clozapine treatment (Riv
a et al. , Mol. Brain. Res. 50, 136-142 (19
97)).

Furthermore, striatal pathological and structural changes have been observed after long-term drug treatment. Studies using laboratory animals have detected a reduction in striatal neuron density and number and size of striatal neurons and neural processes after long-term treatment with haloperidol (Christensen et al., Acta. Ps).
ych. Scand. 46, 14-23 (1970), Jeste et al.
． , Psychopharm. , 106, 154-160 (1992); Mah.
adik et al. , Biol. Psych. , 24, 199-217 (1
988); Nielson et al. , Psychopharm. , 59,
58-89 (1978). These studies implied that neuroleptics may have neurotoxic effects on the striatum, which explains the side effects induced by neuroleptic administration.

The above studies have examined the expression of several individual target genes, but in brain regions, striatum and nucleus accumbens that are believed to be critically involved in the action of neuroleptic drugs. A comprehensive study of the effects of neuroleptic drugs on long-term gene expression in E. coli has not been performed. Therefore, the number and identity of differentially expressed genes in these tissues after short-term and long-term treatment with neuroleptic drugs is unknown. further,
No comprehensive study of the difference between striatal mRNA expression induced by representative neuroleptic drugs and atypical neuroleptic drug expression has been performed.
Such comparative studies will identify genes that control the antipsychotic effects of neuroleptic drugs, against genes involved in unwanted side effects associated with these drugs. This information will facilitate the development of antipsychotic therapies that target specific actions of neuroleptics or selectively inhibit proteins that cause motor side effects.

In addition, systematic characterization will allow identification of genes responsible for neuropathology associated with neuropsychiatric disorders, such as psychosis, bipolar disorder, and behavior associated with addiction. Let's do it. This information not only provides insight into what is the basis of mental illness, but can reveal pathways of mechanism of action of antipsychotics. Thus, the identification of potentially harmful gene products is important for identifying molecules that are useful as diagnostic markers of neuropathology. Moreover, the identification of potentially harmful gene products is important in identifying molecules that are amenable to pharmacological intervention. Systematic characterization also allows the identification of valuable molecules involved in neuroprotective status. Identification of such beneficial products will lead to the development of pharmacological agents useful in the treatment of neuropsychiatric disorders. In addition, the identification of harmful and beneficial products may open a new line of research aimed at improving the symptoms associated with neuropsychiatric disorders.

[0016] The study analyzed multiple gene expression patterns over time following clozapine drug treatment, and in order to compare expression patterns, PCR-based Total Gene Expression Analysis (T).
OGA) method. Genes regulated by clozapine treatment were tested in haloperidol-treated animals for comparative analysis.

SUMMARY OF THE INVENTION The study is to analyze a large number of gene expression patterns over time following clozapine drug treatment, and to compare expression patterns, PCR-based total gene expression analysis (Total Gene Expression Analysis). (T
OGA) method. Genes regulated by clozapine treatment were tested in haloperidol-treated animals for comparative analysis. TOGA analysis identified several genes whose expression was altered in response to clozapine and / or haloperidol administration in mouse brain. In particular, the TOGA system has been used to investigate how genes are regulated by atypical neuroleptic drugs such as clozapine in the striatum and nucleus accumbens. These studies have identified proteins and genes that are regulated by the treatment of atypical drugs. Moreover, these studies have identified at least one gene that is differentially regulated by representative and atypical drugs.

The study also examined the expression patterns of neuroleptic-regulated genes in various regions of the brain. Among other things, these studies are particularly useful for determining genes associated with antipsychotic activity as compared to genes associated with extrapyramidal side effects, which information facilitates the development of improved antipsychotic therapies. The identified neuroleptic-controlled molecules are useful in therapeutic and diagnostic applications in the treatment of various neuropsychiatric disorders, such as psychosis, bipolar disorder, and behavior associated with addiction. Such molecules are also useful as probes, as described by their size, partial nucleotide sequences, and characteristic regulatory patterns associated with neuroleptic drug administration.

The present invention provides novel polynucleotides and encoded polypeptides.
Further, the invention relates to vectors, host cells, antibodies, and recombinant methods for producing polynucleotides and polypeptides. One aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide selected from the following group: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, sequence number 7, sequence number 8, sequence number 9, sequence number 10, sequence number 11, sequence number 12, sequence number 13, sequence number 14, sequence number 15, sequence number 16, sequence number 17, sequence number 18, SEQ ID NO: 19, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 6
6, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71
, SEQ ID NO: 72, and SEQ ID NO: 107. Also, an isolated nucleic acid molecule comprising a polynucleotide that is at least 95% identical to any one of these isolated nucleic acid molecules, and any one of these isolated nucleic acid molecules under stringent conditions. Also provided is an isolated nucleic acid molecule that is at least 10 bases in length hybridizable to. Any one of these isolated nucleic acid molecules can contain sequential nucleotide deletions at either the 5'-end or the 3'-end. Further provided are recombinant vectors containing any one of these isolated nucleic acid molecules and recombinant host cells containing any one of these isolated nucleic acid molecules. Also provided is the gene corresponding to the cDNA sequence of any one of these isolated nucleic acid molecules.

Another aspect of the invention provides an isolated polypeptide encoded by a polynucleotide selected from the following group: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4. Sequence number 5, sequence number 6, sequence number 7, sequence number 8, sequence number 9, sequence number 10, sequence number 11, sequence number 12, sequence number 13, sequence number 14
, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19,
Sequence number 49, sequence number 50, sequence number 51, sequence number 52, sequence number 57, sequence number 58, sequence number 59, sequence number 60, sequence number 61, sequence number 62, sequence number 63, sequence number 64, sequence number 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, and SEQ ID NO: 107. An isolated nucleic acid molecule encoding any of these polypeptides,
An isolated nucleic acid molecule encoding a fragment of any of these polypeptides,
Also provided are isolated nucleic acid molecules encoding the polypeptide epitopes of any of these polypeptides, as well as isolated nucleic acid molecules encoding species homologues of any of these polypeptides. Another aspect of the invention provides an isolated polypeptide of SEQ ID NO: 109. Another aspect of the invention provides an isolated polypeptide of SEQ ID NO: 110. Preferably, any one of these polypeptides has biological activity. Any one of the isolated polypeptides has a C-terminus or N
It may also include sequential terminal amino acid deletions. Further provided are recombinant host cells that express any one of these isolated polypeptides.

Yet another aspect of the invention comprises an isolated antibody that specifically binds to an isolated polypeptide encoded by a polynucleotide selected from the following group: SEQ ID NO: 1, sequence No. 2, SEQ ID No. 3, Sequence No. 4, Sequence No. 5, Sequence No. 6
, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, sequence No. 19, SEQ ID No. 49, SEQ ID No. 50, SEQ ID No. 5
1, SEQ ID NO: 52, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60
, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65,
Sequence number 66, sequence number 67, sequence number 68, sequence number 69, sequence number 70, sequence number 71, sequence number 72, and sequence number 107. Yet another aspect of the invention includes an isolated antibody that specifically binds to the isolated polypeptide of SEQ ID NO: 109. Yet another aspect of the invention includes an isolated antibody that specifically binds to the isolated polypeptide of SEQ ID NO: 110. The isolated antibody may be a monoclonal antibody or a polyclonal antibody.

Another aspect of the invention is preventing a medical condition, such as a neuropsychiatric disorder, comprising administering to a mammalian subject a therapeutically effective amount of a polypeptide of the invention or a polynucleotide of the invention, Methods are provided for treating, modulating, or ameliorating. In a preferred aspect, a method of preventing, treating, modulating or ameliorating schizophrenia is provided. In another preferred embodiment, a method of preventing, treating, modulating or ameliorating bipolar disorder is provided. In yet another preferred embodiment, a method of preventing, treating, modulating or ameliorating behavior associated with addiction is provided.

Another aspect of the invention provides an isolated antibody that specifically binds to the isolated polypeptide of the invention. A preferred embodiment of the invention provides a method for preventing, treating, modulating or ameliorating a medical condition, such as a neuropsychiatric disorder, comprising administering to a mammalian subject a therapeutically effective amount of an antibody. In a preferred aspect, a method of preventing, treating, modulating or ameliorating schizophrenia is provided. In another preferred embodiment, a method of preventing, treating, modulating or ameliorating bipolar disorder is provided.
In yet another preferred embodiment, a method of preventing, treating, modulating or ameliorating behavior associated with addiction is provided.

Another aspect of the invention provides a method of diagnosing a pathological condition, or susceptibility to a pathological condition, in a subject. The method comprises determining the presence or absence of a mutation in the polynucleotide of the invention. Pathological conditions such as neuropsychiatric disorders, or susceptibility to pathological conditions, are diagnosed based on the presence or absence of mutations. In a preferred aspect, a method for diagnosing schizophrenia is provided. In another preferred embodiment, a method for diagnosing bipolar disorder is provided. In yet another aspect, methods of preventing, treating, modulating or ameliorating habit-related behaviors are provided.

In another aspect of the present invention, there is also provided a method for diagnosing a pathological condition such as a neuropsychiatric disorder in a subject. Particularly preferred embodiments include diagnostic methods for schizophrenia and bipolar disorder. This method comprises detecting a change in the expression of the polypeptide encoded by the polynucleotide of the invention, wherein the presence of the change in the expression of the polypeptide is indicative of a pathological condition or a pathological condition. . The change in expression can be an increase in expression or a decrease in expression. In a preferred embodiment, the first biological sample is obtained from a patient suspected of having a neuropsychiatric disorder, such as schizophrenia, bipolar disorder, or addiction-related behaviour, and a second biological sample from a suitable reference source. A sample of is obtained. The amount of at least one polypeptide encoded by the polynucleotide of the invention is determined in the first and second samples.
The amount of the polypeptide in the first and second samples is determined. A patient is diagnosed with a neuropsychiatric disorder if the amount of the polypeptide in the first sample is greater than or less than the amount of the polypeptide in the second sample.

Another aspect of the invention provides a method of identifying a partner that binds to a polypeptide of the invention. A polypeptide of the invention is contacted with a binding partner and it is determined whether this binding partner affects the activity of the polypeptide.

Yet another aspect of the invention is a method of identifying the activity of an expressed polypeptide in a biological assay. The polypeptides of the present invention are expressed intracellularly and isolated. The expressed polypeptide is tested for activity in a biological assay and the activity of the expressed polypeptide is identified based on the test results.

Yet another aspect of the present invention provides a sufficiently pure isolated DNA molecule suitable for use as a probe of a gene regulated in neuropsychiatric disorders, said D
The NA molecule is selected from the group consisting of the DNA molecules represented by the following SEQ ID NOs: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, sequence SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 49 , SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 6
5, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70
, SEQ ID NO: 71, SEQ ID NO: 72, and SEQ ID NO: 107.

Yet another aspect of the invention provides a kit for detecting the presence of a polypeptide of the invention in a mammalian tissue sample. The kit comprises a mammalian protein encoded by a gene corresponding to the polynucleotide of the present invention or a primary antibody immunoreactive with a polypeptide encoded by the polynucleotide, in an amount sufficient for at least one assay, and an appropriate amount. Including packaging material. The kit further comprises a secondary antibody that binds to the primary antibody. Secondary antibodies are enzymes, radioisotopes, fluorescent substances,
It can be labeled with colloidal metals, chemiluminescent substances, luminescent substances, or bioluminescent substances.

Another aspect of the invention comprises a polynucleotide of the invention, or a fragment thereof, having at least 10 contiguous bases in an amount sufficient for at least one assay, and suitable packaging material, A kit for detecting the presence of a gene encoding a protein is provided.

Yet another aspect of the invention provides a method for detecting the presence of a nucleic acid encoding a protein in a mammal. The polynucleotide of the present invention or a fragment thereof having at least 10 contiguous bases is hybridized with a sample nucleic acid. The presence of hybridized product is detected.

Detailed Description of the Preferred Embodiment Definitions The following definitions are provided to facilitate understanding of the terms used herein.

In the present invention, “isolated” means that the object has been removed from its original environment (eg, the natural environment if it is naturally occurring), and thus “human "By hand" is modified from its natural state. For example, an isolated polynucleotide can be part of a vector or a component of an entity, or can be contained within a cell and further “isolated” because the vector, component of the entity, or particular cell is This is because it is not the original environment of the polynucleotide.

In the present invention, a “secreted” protein does not include a protein that can be directed to the endoplasmic reticulum (ER), secretory bleb, or extracellular space as a result of the signal sequence, as well as not necessarily a signal sequence. Refers to proteins released into the extracellular space. When the secreted protein is released into the extracellular space, the secreted protein undergoes extracellular processing to become the "mature" protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.

In the present specification, “polynucleotide” refers to SEQ ID NOs: 1-19; 49-52; 57.
-72; and 107 having a nucleic acid sequence contained in 107. For example, polynucleotides include 5'and 3'untranslated sequences, coding regions with or without signal sequences, secretory protein coding regions, and fragments, epitopes, domains,
And a variant of the nucleic acid sequence, can include all or part of the full-length cDNA nucleic acid sequence. Further, as used herein, "polypeptide" refers to a molecule having a translated amino acid sequence derived from a polynucleotide in its broadest sense.

The “polynucleotide” of the present invention refers to SEQ ID NOs: 1-19; 49-52; 57-7.
Polynucleotides capable of hybridizing to the sequences contained in 2 and 107 or their complementary sequences, or cDNA under stringent hybridization conditions are also included. "Stringent hybridization conditions"
Is 50% formamide, 5xSSC (750 mM sodium chloride, 75 mM
Sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, 20 μg / ml denaturation, sheared salmon sperm DN
After incubation at 42 ° C in A overnight, the filter is washed in 0.1xSSC at 65 ° C.

Further considered are nucleic acid molecules that hybridize to the polynucleotides of the invention under lower stringency hybridization conditions.
Changes in hybridization stringency and signal detection are primarily achieved by manipulation of formamide concentration (lower percent formamide leads to lower stringency); salt concentration, or temperature. For example,
Lower stringency conditions are 6xSSPE (20xSSPE = 3M sodium chloride; 0.2M sodium dihydrogen phosphate; 0.02M EDTA, pH 7.
4), 0.5% SDS, 30% formamide, 100 μg / ml salmon sperm blocking DNA in a solution at 37 ° C. after overnight incubation at 50 ° C.
Washing with 1 × SSPE, 0.1% SDS at room temperature. Furthermore, to obtain lower stringency, washing after stringent hybridization can be done at higher salt concentrations (eg 5xSSC).

Here, variations on the above conditions may be achieved by the addition and / or substitution of some blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent,
Includes BLOTTO, heparin, denatured salmon sperm DNA, proprietary proprietary composition. Due to compatibility issues, the addition of certain blocking reagents may require modification of the above hybridization conditions.

Of course, a polynucleotide that hybridizes only to the poly A sequence (3'-terminal poly A + region of any of the cDNAs shown in the sequence listing) or the complementary stretch T (or U) residue Would not be included in the definition of "polynucleotide" because such a polynucleotide may be attached to any nucleic acid sequence containing a poly (A) stretch or its complement (eg, virtually any double-stranded cDNA It will hybridize (even in clones).

The polynucleotide of the present invention may be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, the polynucleotide can be single and double stranded DNA, DNA that is a mixture of single and double stranded portions, single and double stranded RNA, and RNA that is a mixture of single and double stranded portions, single stranded. It can be composed of a hybrid component consisting of DNA and RNA, or more typically a mixture of double-stranded or single- and double-stranded portions. Further, the polynucleotide can be composed of RNA or DNA or a triple moiety composed of both RNA and DNA. A polynucleotide may include one or more modified bases or DNA or RNA backbones modified for stability or other reasons. "Modified" bases include unusual bases such as tritylated bases and inosine. Various modifications are made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms.

The polypeptides of the invention may contain amino acids other than the 20 gene-encoded amino acids, which consist of amino acids linked to each other by peptide bonds or modified peptide bonds, ie, peptide isosteres. The polypeptide may be modified by natural processes such as post-translational modification or by chemical modification techniques known to those skilled in the art. Such modifications are well documented in basic texts and more detailed books as well as in the large research literature. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type of modification may be present in the same or varying degrees at several places in a polypeptide. Also, a given polypeptide may contain many types of modifications. The polypeptide is, for example, ubiquitination (ub
It may or may not be branched as a result of the initiation, but may be cyclic. Cyclic, branched, branched cyclic polypeptides may be the result of posttranslational natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP ribosylation, amidation, flavin covalent bond, heme molecule covalent bond, nucleotide or nucleotide derivative covalent bond, lipid or lipid derivative covalent bond, phosphatidylinositol covalent bond. Bond, crosslink,
Cyclization, disulfide bond formation, dimethylation, covalent crosslink formation, cysteine formation, pyroglutamic acid formation, formation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation , Myristoylation, oxidation,
Pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation
), Sulfation, addition of amino acids to proteins by transfer RNA such as arginylation and ubiquitination (for example, TECreghton, Proteins-Structure and Molecular Properties, 2nd Ed., W.
H. Freeman and Company, New York (1993); BCJohnson, Ed., Posttranslatio
nal Covalent Modification Of Proteins, Academic Press, New York, pgs. 1-
12 (1983); Seifter et al., Meth. Enzymol, 182: 626-647 (1990); Rattan et a.
l., Ann. NYAcad.sci. 663: 48-62 (1992)).

A “biologically active polypeptide” is not necessarily identical to a polypeptide of the invention in its mature form, with or without a dose dependence, as measured by a particular biological method. It refers to a polypeptide that may be absent, but exhibits similar activity. If it is dose-dependent, then it need not be identical to that of the polypeptide of the invention, but is nearly as dose-dependent in some activity as compared to the polypeptide of the invention (ie the candidate polypeptide is It will exhibit greater activity, or activity that is not more than about 25-fold, preferably not more than about 10-fold, and most preferably not more than about 3-fold against the polypeptides of the invention).

The translated amino acid sequence begins with methionine and is identified, although other reading frames can be readily translated using known molecular biology techniques. Polypeptides produced by translation of these other open reading frames are specifically contemplated by the present invention.

SEQ ID NO: 1-19; 49-52; 57-72 and 107 and SEQ ID NO: 1-
The translations of 19; 49-52; 57-72 and 107 are sufficiently accurate and suitable for a variety of uses well known to those of skill in the art and described further below. These probes also hybridize to nucleic acid molecules in biological samples, thus enabling the various forensic and diagnostic methods of the invention. Similarly, SEQ ID NOs: 1-19; 49-52;
Polypeptides identified from translations of 57-72 and 107 were identified by the identified cDNA.
It may be used to produce antibodies that specifically bind to the secreted protein encoded by the A clone.

The DNA sequence generated by the sequencing reaction may contain sequencing errors. Errors exist as misidentified nucleotides in the resulting DNA sequence, or as insertions or deletions of nucleotides. Misinserted or deleted nucleotides cause a frameshift in the reading frame of the predicted amino acid sequence. In such cases, the resulting DNA sequence is
The predicted amino acid sequence differs from the true amino acid sequence even though it is 99.9% or more homologous to the sequence (eg, a single base insertion or deletion in the open reading frame of more than 1000 bases).

The present invention also relates to the genes corresponding to SEQ ID NOs: 1-19; 49-52; 57-72 and 107, and translations of SEQ ID NOs: 1-19; 49-52; 57-72 and 107. Corresponding genes can be separated by known methods using the sequence information disclosed herein. Such methods include the preparation of probes and primers from the disclosed sequences and identification and amplification of the corresponding gene from a suitable source of genomic material.

The present invention also provides interspecies homologues. Interspecies homologs may be isolated and identified by making appropriate probes or primers from the sequences provided herein and screening for an appropriate nucleic acid source for the desired homolog.

The polypeptides of the present invention can be prepared by any suitable method. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means of preparing such polypeptides are known to those of skill in the art.

The polypeptide may be in the form of a secreted protein, including the mature form, or may be part of a larger protein such as a fusion protein (
See below). It is often advantageous to include secretory or leader sequences, prosequences, additional amino acid sequences that include sequences that aid in purification such as multiple histone residues, or additional sequences for stability during recombinant production. .

The polypeptides of the present invention are preferably provided in isolated form and are suitable and well purified. Among the polypeptides, those produced recombinantly (including secretory polypeptides) can be sufficiently purified by the one-step method described in Smith and Johnson, Gene 67: 31-40 (1988). The polypeptides of the present invention can also be purified from natural or recombinant materials using the antibodies of the present invention raised against secreted proteins by known methods.

Signal Sequence Whether or not the protein has a signal sequence and the cleavage point of that sequence (cleavag
A method of predicting e point) is available. For example, McGeoch's method uses a short N
Information from the terminal charged portion and the fully uncharged portion of the intact (uncleaved) protein is used (Virus Res., 3: 271-286 (1985)). The von Heinje method uses information from residues around the cleavage site, typically residues -13 to +12, where +1 indicates the amino terminus of the secreted protein (Nucleic acids Res.
, 14: 4683-4690 (1986)). Therefore, a signal sequence and a mature sequence can be identified from the determined amino acid sequence.

In this case, the determined amino acid sequence of the secreted polypeptide is a computer program called Signal P (Nielsen et al., Protein) that predicts the intracellular localization of the protein based on the amino acid sequence. Engineering, 10: 1-6 (1997))
Analyzed by Part of the computer prediction of this localization is McGeoch and von.
Heinje's method was incorporated.

As will be understood by one of ordinary skill, the cutting site can vary from organism to organism and cannot be predicted with perfect certainty. Therefore, in the present invention, a secretory polypeptide having a sequence corresponding to the translation of translation numbers 1-19, which starts within 5 residues of the predicted cleavage point (that is, + or -5 residues)
Provide one with ends. Similarly, it will be appreciated that, in some cases, cleavage of the signal sequence from the secreted protein is not completely homogenous, resulting in one or more secreted species. These polypeptides and polynucleotides encoding these polypeptides are contemplated by the invention.

Furthermore, it can be said that the signal sequences identified by the above analysis are not necessarily predictive of naturally occurring signal sequences. For example, the naturally occurring signal sequence may be upstream of the predicted signal sequence. However, the predicted signal sequence could direct the secreted protein to the endoplasmic reticulum. These polypeptides and polynucleotides encoding these polypeptides are contemplated by the invention.

Variants of Polynucleotides and Polypeptides “Variant” refers to a polynucleotide or polypeptide that differs from the polynucleotide or polypeptide of the invention but retains its basic properties. Variants are generally similar overall and in many regions are consistent with the polynucleotides or polypeptides of the invention.

“Identity” has its own meaning as understood by those of ordinary skill in the art and is calculated using published techniques. (Lesk, AM, Ed., Computational Molecular
Biology, Oxford University Press, New York, (1988); Smith, DW, Ed, Bi
ocomputing: Informatics And Genome Projects, Academic Press, New York, (
1993); Griffin, AM, and Griffin HG, Eds., Computer Analysis Of Seque
nce Data, Part I, Humana Press, New Jersey, (1994); von Heinje, G., Seque
nce Analysis In Molecular Biollogy, Academic Press, (1987); Gribskov, M.
and Devereux, J., Eds., Sequence Analysis Primer, M. Stockton Press, Ne
See w York, (1991). The term “homology” is well known to the person skilled in the art, among the many ways in which to determine the homology between two polynucleotide or polypeptide sequences. (Carillo, H., and Lipton, D., SIAM J. Applied Math., 48: 1073 (19
88). ) Methods commonly used to determine identity and similarity between two sequences are described in Martin J. Bishop, eds. "Guide to Huge Computers," Academic Press, S
an Diego, (1994), and Carillo, H., and Lipton, D., SIAM J. Applied Math,
48: 1073 (1988), but is not limited thereto. The method for aligning polynucleotides and polypeptides is described in GCG program package (Devereux, J., et al., Nuc. Acids Res. 12 (1): 387 (1984)), BLASTP, BLA.
STN, FASTA (Atschul, SF et al., J. Molec. Biol. 215: 403 (1990), Bestfit.
program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Geneti
cs Computer Group, University Rresearch Park, 575 Scieince Drive, Madiso
n, WI 53711 (Smith and Waterman, Advances in Applied Mathematics 2: 482-48
9 (using local homology in 1981)).

Specific sequences can be referenced, for example, using any sequence alignment program.
When determining whether to be 95% homologous to a (reference) sequence, the percentage of homology is calculated over the entire length of the reference polynucleotide, allowing a homology gap of up to 5% of the total number of nucleotides in the reference polynucleotide. Parameters are set as follows.

A preferred method for determining the best overall match between a query sequence (the sequence of the invention) and a subject sequence, also referred to as global sequence alignment, is described by Brutlag et al. Algorithm (Comp, App. Biosci., 6: 237-245 (199
It can be determined using the FASTDB computer program based on 0)).
The term "sequence" includes nucleotide and amino acid sequences. In a sequence alignment, both the query and the target sequence are nucleotide sequences or both are amino acid sequences. The global sequence alignments are expressed as percent homology. The preferred parameters used to calculate percent homology in a FASTDB search of DNA sequences are: Matrix = Unitary, k-tuple = 4, Misma
tch Penalty = 1, Joining Penalty = 30, Randomization Group Length = 0, and Cu
toff Score = 1, Gap Penalty = 5, Gap Size Penalty 0.05, and Window Size = 500
Or, the shorter of the number of query sequences of nucleotide bases. The preferred parameters used to calculate the percent homology and similarity of amino acid alignments are: Matrix = PAM150, k-tuple = 2, Mismatch Penalty = 1, Joining Penalt
y = 20, Randomization Group Length = 0, Cutoff Score = 1, Gap Penalty = 5, Gap Si
ze Penalty = 0.05 and Window Size = 500 or the query sequence length of amino acid residues, whichever is shorter.

By way of example, a polynucleotide having a nucleotide sequence that is at least 95% “homologous” to the sequences contained in SEQ ID NOs: 1-19; 49-52; 57-72 and 107 is No. 1-19; 49-52; 57-7
2 and 107 or homologous to a sequence contained in the cDNA, provided that the polynucleotide sequence contains up to 5 point mutations per 100 nucleotides of full length (not only within a particular 100 nucleotide portion) It means good. In other words, SEQ ID NOs: 1-19; 49-52; 5
To obtain a polynucleotide having a nucleotide sequence at least 95% homologous to the sequences contained in 7-72 and 107, SEQ ID NOs: 1-19; 49-5
2; 57-72 and 107 or up to 5% of the nucleotides in the sequence contained in the cDNA may be deleted, inserted or replaced by other nucleotides. These changes may occur anywhere in the entire polynucleotide.

A further aspect of the invention is SEQ ID NOS: 1-19; 49-52; 57-72 and 1.
At least 80% homology to the sequence contained in 07, more preferably at least 90% homology, most preferably at least 95%, 96%, 97%,
It includes polynucleotides with 98% or 99% homology. of course,
Due to the degeneracy of the genetic code, those of ordinary skill in the art will be at least 85%, 90%
, 95%, 96%, 97%, 98%, or 99% homologous polynucleotides are homologous to the amino acid sequences contained in the translations of SEQ ID NOs: 1-19; 49-52; 57-72 and 107. You will immediately recognize that they will encode different polypeptides.

Similarly, a polypeptide that has an amino acid sequence that has “homology” to the reference polypeptide, eg, has at least 95% “homology,” is one in which the amino acid sequence of the polypeptide is homologous to the reference polypeptide, provided that the polypeptide sequence is It is contemplated that up to 5 amino acid mutations may be included in every 100 amino acids of the full length reference polypeptide. That is, in order to obtain a polypeptide having an amino acid sequence at least 95% homologous to the reference amino acid sequence, up to 5% of the amino acid residues of the reference sequence may be deleted or substituted with another amino acid, or Multiple amino acids up to 5% of all amino acid residues in may be inserted in the reference sequence. These variations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence, or anywhere between those terminal positions, whether interspersed separately within the residues in the reference sequence, It may occur within one or more adjacent groups in the reference sequence.

A further aspect of the invention is at least 80% homology, more preferably at least 90% homology to the amino acid sequences involved in the translation of SEQ ID NOs: 1-19; 49-52; 57-72 and 107. Homology of, and most preferably at least 9
It includes polypeptides with 5%, 96%, 97%, 98% or 99% homology. Preferably, the above polypeptides should exhibit at least one biological activity of the protein of the invention.

In a preferred embodiment, the polypeptides of the present invention are SEQ ID NOs: 1-19; 49.
-52; preferably 90% homology, more preferably at least 95% homology, and even more preferably at least 96%, 97%, 98% to the amino acid sequences involved in the translation of 57-72 and 107. , Or alternatively, a polypeptide having 99% homology.

Variants include alterations in the coding regions, non-coding regions, or both.
Particularly preferred are those that result in silent substitutions, additions or deletions,
However, it is a variant of a polynucleotide containing modifications that do not alter the properties or activity of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Furthermore, mutants in which 5-10, 1-5 or 1-2 amino acids are substituted, deleted or added in any combination are also preferable. Variants of polynucleotides are produced for a variety of reasons. For example, a variant of a polynucleotide may be made to optimize codon expression for a particular host, ie the codons in human mRNA may be changed to those favored by a bacterial host such as E. coli.

Naturally occurring variants are referred to as “allelic variants” and refer to one of several different forms of a gene that occupy a chromosomal location in one organism (Lewin, B. , Edited by Genes II, John Wiley & Sons, New York (1985)). These allelic variants can be mutated at the polynucleotide and / or polypeptide level. Also, non-naturally occurring variants may be made by mutagenesis techniques or direct synthesis.

Well-known protein engineering and recombinant DNA techniques may be used to generate variants which improve or alter the properties of the polypeptides of the present invention. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of a secreted protein without significantly losing biological activity. Ron et al. Reported a mutant of KGF protein having heparin-binding activity even after deletion of 3, 8 or 27 amino terminal amino acid residues (J. Biol. Chem .., 268: 2984-2988 (1993). ). Similarly, gamma interferon showed up to 10-fold higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein (Dobeli et al., J. Biothchnology. 7: 199.
-216 (1988)).

In addition, ample evidence indicates that the variants often retain biological activity comparable to that of naturally occurring proteins. For example, Gayle et al. Performed extensive mutational analysis of the human cytokine IL-1a (J. Biol. Chem., 268: 22.
105-22111 (1993)). They use random mutations to generate over 3,500 independent IL-1a with an average of 2.5 amino acid mutations per variant over the entire length of the molecule.
A mutant was created. Multiple mutations were examined at all possible amino acid positions. The researchers concluded, "we were able to modify most molecules with little effect on either binding or biological activity." (Gayle et al., (1993
), See overview. In fact, of the over 3,500 nucleotide sequences examined, only 23 unique amino acid sequences produced proteins that differed significantly in activity from the wild type.

Furthermore, even if one or more amino acids are deleted from the N-terminus or C-terminus of the polypeptide, resulting in modification or disappearance of one or more biological activities, other biological activities are still retained. You may be drunk. For example, the activity of the deletion mutant to induce and / or bind to an antibody that recognizes a secreted form is retained when less than the majority of the secreted forms are removed from the N- or C-termini. Often. Whether a particular polypeptide lacking the N- or C-termini of a protein retains such immunogenetic activity is described herein and in other references known to those of skill in the art. It can be easily determined by conventional methods.

Accordingly, polypeptide variants that exhibit sufficient biological activity are further included in the present invention. Such variants include deletions, insertions, inversions, repeats, and substitutions chosen so that activity is not significantly affected by well-known general rules. For example, for teaching how to make phenotypically silent amino acid substitutions, see Bowie,
et al., Science, 247: 1306-1310 (1990), in which the authors state that there are two main strategies for studying tolerance to amino acid sequence changes. .

The first strategy uses the tolerance of amino acid substitutions by natural selection during evolution. Conserved amino acids are identified by comparing amino acid sequences between different species. These conserved amino acids may be important for protein function. In contrast, the amino acid sites that have been allowed for substitution in natural selection indicate that these sites are not essential for protein function. Therefore,
The sites that allow amino acid substitutions could be modified while retaining the biological activity of the protein.

The second strategy is to introduce an amino acid mutation into a specific site of the cloned gene using genetic engineering to identify a region essential for protein function. For example, site-directed mutations or alanine-scanning mutations (introduction of one alanine mutation for each residue in the molecule) can be used (Cu
nningham and Wells, Science, 244: 1081-1085 (1989)). The resulting mutant molecule can then be subjected to an assay for biological activity.

According to Bowie et al., These two strategies have revealed that proteins are surprisingly permissive for amino acid substitutions. The authors also show which amino acids are more likely to be tolerated at certain amino acid sites within the protein. For example, the most embedded amino acid residues (within the tertiary structure of the protein) require non-polar side chains and the nature of surface side chains is generally conserved. Furthermore, the conservative amino acid substitutions allowed are the substitution of aliphatic or hydrophilic amino acids Ala, Val, Leu, Ile; the substitution of hydroxyl residue Ser, Thr; the acidic residue Asp, Glu.
Substitution of amide residues Asn, Gln; substitution of basic residues Lys, Arg, His; substitution of aromatic residues Phe, Tyr, Trp; and small-sized amino acids Ala, Ser, Thr, Met, G
ly replacements, and so on.

In addition to conservative amino acid substitutions, variants of the invention include (i)
Substitution with one or more non-conserved amino acid residues in which the substituted amino acid residues are encoded or not encoded by the genetic code, or (ii)
Substitution by one or more amino acid residues having a substituent, or (iii) fusion of the mature polypeptide of the present invention with another compound, for example a compound which improves the stability and / or solubility of the polypeptide ( (Eg polyethylene glycol), or (iv) a fusion with an additional amino acid of a polypeptide of the invention, eg IgG Fc
Binding site peptides, or leader or secretory sequences, or sequences that facilitate purification. Such variant polypeptides are considered within the purview of those skilled in the art given the teachings herein.

For example, variants of the polypeptide that include substitution of charged amino acids for other charged or neutral amino acids may produce proteins with improved properties, such as reduced aggregation. As is well known, aggregation of pharmaceutical compositions reduces activity and increases clearance due to immunogenetic activity of the aggregate (eg Pinckard et al., Clin. Exp. Immunol. 2: 331-340. (1967); Robbins et al.,
Diabetes, 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug
Carrier Systems, 10: 307-377 (1993)).

Polynucleotides and Polypeptide Fragments In the present invention, “polynucleotide fragments” are short polynucleotides and are included in the sequences shown in SEQ ID NOs: 1-19; 49-52; 57-72; and 107. It has a nucleic acid sequence. This short nucleotide fragment is preferably at least about 15 nucleotides in length, more preferably at least about 20 nucleotides, even more preferably at least about 30 nucleotides, and even more preferably at least about 40 nucleotides. The fragment of "at least 20 nucleotides in length" refers to, for example, SEQ ID NOs: 1-19; 49-52;
7-72; and 20 or more contiguous bases from the cDNA sequence contained in the sequence shown in 107. These nucleotide fragments are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments (eg 50, 150, and even more nucleotides) are preferred.

Further, typical examples of the polynucleotide fragment of the present invention include, for example, nucleotide numbers of about 1-50, 51-100, 101-150, 151-200, 201-2.
50, 251-300, 301-350, 351-400, 401-450 to SEQ ID NOs: 1-19; 49-52; 57-72; Here, "about" includes the specifically listed ranges, and is increased or decreased by a small number (5, 4, 3, 2, 1) nucleotides at either or both ends. Preferably, these fragments encode a biologically active polypeptide.

In the present invention, “polypeptide fragment” means SEQ ID NOs: 1-19; 49-52.
57-72; and 107, which are short amino acid sequences involved in the translation. Protein fragments may be "free-standing," or comprised within a larger polypeptide, which fragments most preferably form part or region of the polypeptide as a stretch of site. Representative examples of polypeptide fragments of the invention include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, or 61 to the end of the coding region. In addition, the polypeptide fragment has a length of about 2
It can be 0, 30, 40, 50, or 60 amino acids. here,"
"About" includes the specifically recited ranges, with more or less a few (5,4,3,2,1) amino acids at either or both ends.

Preferred polypeptide fragments include secreted proteins as well as mature forms. Further preferred polypeptide fragments include, from the amino or carboxy terminus,
Alternatively, it includes a secretory protein or a mature form in which residues are consecutively deleted from both. For example, any number of amino acids in the range 1-60 can be deleted from the amino terminus of either the secreted polypeptide or the mature form. Similarly, 1-30
Can be deleted from the carboxy terminus of either the secreted polypeptide or the mature form. Furthermore, any combination of the above amino or carboxy terminal deletions is preferred. Similarly, polynucleotide fragments encoding these polypeptide fragments are also preferred.

Also preferred are polypeptide and polynucleotide fragments characterized by structural or functional domains, such as fragments comprising: alpha helices and alpha helix forming regions, beta sheets or beta sheet forming regions, turns. And turn forming regions, coils and coil forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface forming regions, substrate binding regions, and high antigen index regions. Polypeptide fragments translated from SEQ ID NOs: 1-19; 49-52; 57-72; and 107 that are within the conserved domain are specifically contemplated herein. It also relates to polynucleotide fragments encoding these domains.

Other preferred fragments are bioactive fragments. Biologically active fragments are those that exhibit similar (but not necessarily identical) activities to those of the polypeptides of the present invention. The bioactivity of a fragment can include those with an enhanced desired activity or a reduced undesirable activity.

Epitopes and Antibodies In the present invention, "epitope" refers to a polypeptide fragment having antigenic or immunogenic activity in animals, especially humans. A preferred embodiment of the invention relates to a polypeptide fragment containing an epitope, as well as a polynucleotide encoding this fragment. The region of the protein molecule to which the antigen can bind is defined as the "antigen epitope". In contrast, an "immunogenic epitope" is defined as the part of the protein that elicits an antibody response (eg Geysen et al., Proc. Natl. Acad. Sci. USA, 81.
: 3998-4002 (1983)).

Fragments which function as epitopes may be generated by any conventional method (eg Houghten, RA, Proc. Natl. Acad. Sci. USA, 82: 5131-5135 (198).
See 5). It is also described in US Pat. No. 4,631,211).

In the present invention, preferably the antigenic epitope contains a sequence of at least 7, more preferably at least 9, and most preferably between about 15 and about 30 amino acids. Antigenic epitopes are useful in the production of antibodies, including monoclonal antibodies, which specifically bind to the epitope (eg Wilson et al., Cell,
37: 767-778 (1984); Sutcliffe, JG et al., Science, 219: 660-666 (1983)).

Similarly, immunogenic epitopes can be used to raise antibodies according to methods well known in the art (eg, Sutcliffe et al., Supra; Wilson et al., Supra; Chow, M.
Et al., Proc. Natl. Acad. Sci., USA 82: 910-914; and Bittle, FJ et al., J. Ge.
n. Virol., 66: 2347-2354 (1985)). A preferred immunogenic epitope is a secreted protein. In animal systems (eg rabbit or mouse) the immunogenic epitope may be present together with a carrier protein (eg albumin),
Alternatively, the carrier may not be present if it is sufficiently long (at least about 25 amino acids). However, it appears that even an immunogenic epitope containing only 8 to 10 amino acids is sufficient for a denatured polypeptide (eg, on a Western blot) to produce an antibody capable of binding to it with a very small amount of a linear epitope. Has become.

The term “antibody” (Ab) or “monoclonal antibody” (Mab), as used herein, refers to intact molecules and antibody fragments (eg, Fab and F (ab ′)) that are capable of specifically binding to a protein. 2 fragment). Fab and F (ab ') 2 fragments lack the Fc fragment of an intact antibody, are more rapidly cleared from circulation and may have less nonspecific tissue binding than intact antibody (Wahl et al., J. Nucl. Med., 24: 316-325 (1983)). Therefore, these fragments are preferred as are the products of FAB or other immunoglobulin expression libraries. Furthermore, the antibodies of the present invention include chimeric, single chain, and humanized antibodies.

A further aspect is a humanized version of a chimeric antibody, eg a mouse monoclonal antibody. Such humanized antibodies may be prepared by known techniques and have the advantage of low immunogenicity when administered to humans. In some embodiments, the humanized monoclonal antibody comprises various regions of a mouse antibody (or only its antigen binding site).
And a constant region derived from a human antibody. Alternatively, a humanized antibody fragment may contain the antigen binding site of a mouse monoclonal antibody and a variable region fragment from a human antibody (lacking the antigen binding site). Methods for the generation of chimeras and further engineered monoclonal antibodies include those described below:
Riechmann et al. (Nature, 332: 323, 1988), Liu et al. (PNAS, 84: 3439, 1987), Larr.
ick et al. (Bio / Technology, 7: 934, 1989), and Winter and Harris (TIPS, 14
: 139, May 1993).

One method of producing human antibodies is to provide a non-human animal (eg, a transgenic mouse) with a polypeptide translated from a nucleotide sequence selected from SEQ ID NOs: 1-19; 49-52; 57-72; and 107. Including inoculation, whereby SEQ ID NO: 1-
Antibodies to polypeptides translated from nucleotide sequences selected from 19; 49-52; 57-72; and 107 are produced in the animal. Methods have been developed to produce human antibodies in non-human animals. The antibody may be partially human, or preferably fully human. Non-human animals (eg, transgenic mice) into which the genetic material encoding one or more human immunoglobulin chains has been introduced may be used. The transgenic mice may be genetically modified in various ways. Genetic engineering may replace the endogenous immunoglobulin chains in at least a portion (preferably substantially all) of the antibodies produced by the animal upon immunization with human immunoglobulin polypeptide chains. SEQ ID NO: 1-19; 49-52
Also provided herein are antibodies produced by inoculating transgenic animals with a polypeptide translated from a nucleotide sequence selected from: 57-72; and 107.

Mice in which one or more endogenous immunoglobulin genes have been inactivated by various methods have been prepared. The human immunoglobulin gene was introduced into the mouse to replace the inactivated mouse gene. Antibodies produced in animals incorporate human immunoglobulin polypeptide chains encoded by human genetic material introduced into the animal. Examples of techniques for the generation and use of such transgenic animals are found in US Pat.
4,318; 5,569,825; and 5,545,806, which are incorporated herein by reference.

Monoclonal antibodies may be generated by conventional methods, for example by immortalizing spleen cells recovered from transgenic animals after the immunization schedule has been completed. Spleen cells may be fused with myeloma cells to produce hybridomas by conventional methods.

Methods for producing hybridoma cell lines include: of such a transgenic animal a polypeptide translated from a nucleotide sequence selected from SEQ ID NOs: 1-19; 49-52; 57-72; and 107. Inoculation with an immunogen containing at least 7 consecutive amino acid residues; recovery of spleen cells from the immunized animal; generation of hybridoma cells by fusing the recovered spleen cells to a myeloma cell line; : 1-19; 49-52; 57-72; and 107, identifying hybridoma cell lines that produce monoclonal antibodies that bind to the translated polypeptide from a nucleotide sequence selected from: Those hybridoma cell lines, and the monoclonal antibodies produced therefrom, are within the scope of the invention. Monoclonal antibodies secreted by the hybridoma cell line are purified by conventional techniques.

The antibody may be used in an in vitro method or administered in vivo to SEQ ID NO: 1-1.
It may inhibit biological activity induced by a polypeptide translated from a nucleotide sequence selected from 9; 49-52; 57-72; and 107. Disorders caused or exacerbated (directly or indirectly) by the interaction of such polypeptides of the invention with cell surface receptors may be treated. Therapeutic methods reduce the biological activity induced in vivo by a blocking antibody in mammals by a polypeptide translated from a nucleotide sequence selected from SEQ ID NOs: 1-19; 49-52; 57-72; and 107. Administration in an effective amount. For example, long-term administration of neuroleptics may cause unwanted side effects. Administration of antibodies derived from the identified polynucleotides may block the signaling that triggers these side effects. Alternatively, the identified polynucleotide-derived antibodies may selectively inhibit proteins that cause motile side effects.

Also provided herein are conjugates containing a detectable (eg, diagnostic) or therapeutic agent, from SEQ ID NOs: 1-19; 49-52; 57-72; and 107. It binds to antibodies directed against the polypeptide translated from the selected nucleotide sequence. Examples of such agents are well known and include, but are not limited to, diagnostic radionuclides, therapeutic radionuclides, and cytotoxic agents. Complex is in
It also finds use in in vitro or in vivo methods.

Fusion Proteins Any polypeptide of the invention can be used to produce a fusion protein. For example, the polypeptides of the present invention can be used as an antigen tag when fused to a second protein. Antibodies raised against the polypeptides of the invention can be used to indirectly detect a second protein by binding to the polypeptide. Furthermore, since secreted proteins target cellular locations based on trafficking signals, the polypeptides of the invention can be used as targeting molecules when fused to other proteins.

Examples of domains that can be fused to the polypeptides of the invention include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily have to be direct, but may be via a linker sequence.

Furthermore, the fusion protein may be engineered to enhance the characteristics of the polypeptides of the invention. For example, additional amino acids, especially regions of charged amino acids, may be added to the N-
It may be added to the ends to improve stability and durability during purification from host cells or subsequent manipulation and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. Addition of peptide moieties to facilitate manipulation of polypeptides is well known and routine in the art.

Furthermore, the polypeptides (including fragments) of the invention, and in particular the epitopes, may be combined with part of the constant domain of immunoglobulins (IgG) to give chimeric polypeptides. These fusion proteins facilitate purification and in vivo
It shows an increased half-life at. One reported example describes a chimeric protein consisting of the first two domains of human CD-4 polypeptide and various domains of the constant region of the heavy or light chain of a mammalian immunoglobulin (Europe). Patent Publication EP-A 394,827; see Traunecker et al., Nature, 331: 84-86 (1988)).
. Also, fusion proteins having a disulfide-linked dimer structure (by IgG) can bind or neutralize other molecules more efficiently than the monomeric secretory protein or protein fragment alone (Fountoulakis et al., J. Biochem
270: 3958-3964 (1995)).

Similarly, European Patent Publication EP-A-0 464 533 (corresponding Canadian patent is 204586).
9) discloses fusion proteins containing various parts of the constant regions of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc portion of the fusion protein will be of therapeutic and diagnostic benefit and thus may improve eg pharmacokinetic properties (eg European Patent Publication EP-A-0 2
32 No. 262). Alternatively, it is preferred to remove the Fc portion after expressing, detecting and purifying the fusion protein. For example, the Fc protein can interfere with therapy and diagnosis when using the fusion protein as an antigen for immunization. In drug discovery, for example, human proteins (eg hIL-5) have been fused with Fc proteins for the purpose of high information screening assays and antagonists of hIL-5 have been identified (D. Bennett et al., J. Molecular Recognition, 8: 52-58 (1995)
K. Johanson et al., J. Biol. Chem., 270: 9459-9471 (1995)).

In addition, the peptides of the invention can be fused to a marker sequence, eg, a peptide that facilitates purification of the fused polypeptide. In a preferred embodiment, the marker amino acid sequence is a 6 histidine peptide, such as the pQE vector (Qiagen, 9259 E).
It is a tag provided by ton Avenue, Chatsworth, CA, 91311), and many other tags are commercially available. For example, as reported by Gentz et al., The fusion protein can be conveniently purified by 6-histidine (Proc. Natl. Acad. Sci. USA, 86: 8.
21-824 (1989)). Another peptide tag useful for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell, 37.
: 767 (1984)). Other fusion proteins may use the ability of the polypeptides of the invention to target the delivery of biologically active peptides. This may include the concentrated delivery of toxic substances to tumor cells or growth factors to stem cells.

Accordingly, any of these above fusions can be engineered using the polynucleotides or polypeptides of the invention.

Vectors, Host Cells, and Protein Production The present invention also includes vectors, host cells, containing the polynucleotides of the invention.
And to the production of polypeptides by recombinant techniques. The vector may be, for example, a phage, plasmid, viral vector, or retroviral vector. Retroviral vectors may or may not be replication competent. In the latter case, viral propagation generally will occur only in complementing host cells.

The polynucleotide may be linked to a vector containing a selectable marker for growth in a host. Generally, plasmid vectors are introduced in a precipitate, such as a calcium phosphate precipitate, or in complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using a suitable packaging cell line prior to introduction into host cells.

The polynucleotide insert should be operably linked to a suitable promoter, which includes the phage lambda PL promoter, E. coli lac, trp, phoA, and tac promoters, SV40, to name a few.
The early and late promoters and the promoter of the retrovirus LTR. Other suitable promoters are well known to those of skill in the art. The expression construct further contains sites for transcription initiation, termination, and in the transcribed region a ribosome binding site for translation. The coding portion of the transcript expressed by the construct preferably contains first a translation initiation codon and a termination codon (UAA, UGA, or UAG) suitably located at the end of the polypeptide to be translated.

As mentioned, the expression vector preferably comprises at least one selectable marker. These markers include dihydrofolate reductase, G418, or neomycin resistance for eukaryotic cell cultures and tetracycline, kanamycin, or ampicillin resistance genes for cultures in E. coli and other bacteria. Representative examples of suitable hosts include, but are not limited to: bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium; fungal cells such as yeast cells; insect cells, Eg Drosophila S2 and Spodoptera Sf9 cells; animal cells eg CHO, COS, 2
93, Bowes melanoma cells, and plant cells. Suitable media and conditions for the above host cells are well known in the art.

Vectors suitable for use in bacteria include: pQE70, pQE60, and pQ
E-9 (QIAGEN); pBluescript vector, Phagescript vector, pNH8A, PNH16a
, PNH18A, pNH46A (Stratagene Cloning Systems); and ptrc99a, pKK223-
3, pKK233-3, pDR540, pRIT5 (Pharmacia Biotech). Preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTI, and pSG (Stratagene); pSVK3, pBPV, pM.
SG and pSVL (Pharmacia). Other suitable vectors will be readily apparent to those of skill in the art.

The introduction of the construct into the host cells can be performed by calcium phosphate transfection, DEAE dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, or other methods. These methods are described in many standard laboratory manuals, such as Davi.
s et al., “Basic Methods In Molecular Biology”
) ", (1986). In particular, it is contemplated that the polypeptides of the present invention may be expressed by host cells that do not actually contain a recombinant vector.

Currently, there are no specific diagnostic markers that can be used to prevent or delay the onset of the psychotic symptoms of schizophrenia. The polypeptides of the present invention can be used as chromosomal markers for the diagnosis of schizophrenia. The polypeptides of the present invention can be recovered and purified from recombinant cell culture media by well known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography. There are chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Most preferably, it is purified using high performance liquid chromatography (“HPLC”).

Polypeptides of the invention, preferably in secreted form, can also be recovered from: Natural sources, including body fluids, tissues, and cells, either directly isolated or cultured. Products purified from; chemical synthesis products; and products produced by recombinant techniques from prokaryotic or eukaryotic hosts, including bacteria, yeast, higher plants, insects, and mammalian cells. The polypeptides of the invention may or may not be glycosylated, depending on the host used in the recombinant production procedure. Furthermore, the polypeptides of the present invention may include an initial modified methionine residue, in some cases as a result of host-mediated processes. Therefore, as is known in the art, the N-terminal methionine encoded by the translation initiation codon in all eukaryotic cells is generally highly efficiently removed from the protein after translation. Most prokaryotic cells efficiently remove the N-terminal methionine on most proteins, but for some proteins this prokaryotic removal process is inefficient, because the amino acid to which the N-terminal methionine is covalently bound. Depends on the nature of.

Uses of Polvnucleotides Each of the polypeptides identified herein can be used as a reagent in a number of ways. The following description should be considered representative and uses known techniques.

The polynucleotide of the present invention is useful for chromosome identification. Currently, there is a need to identify new chromosomal markers, as chromosomal markers based on actual sequence data (repeated polymorphisms) are so far unavailable. Each polynucleotide of the present invention can be used as a chromosomal marker.

Briefly, the sequence can be mapped to the chromosome by preparing PCR primers (preferably 15-25 bp) from the sequences shown in SEQ ID NOs: 1-19; 49-52; 57-72; and 107. . Primer is a genomic DN using computer analysis
The primers in A can be selected so that they do not span more than one predicted exon. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing a human gene corresponding to SEQ ID NOs: 1-19; 49-52; 57-72; and 107 produce amplified fragments.

Similarly, somatic cell hybrids provide a rapid PCR method for mapping polynucleotides to specific chromosomes. Three or more clones can be assigned in one day using one thermocycler. Furthermore, the semi-localization of the polynucleotide
It can be done with a series of specific chromosomal fragments. Other gene mapping methods that can be used include in situ hybridization, labeled flow-sorted
There is prescreening on the chromosome and preselection by hybridization to construct a chromosome-specific cDNA library.

The precise chromosomal location of the polynucleotide can be determined using fluorescence in situ hybridization (FISH) of metaphase chromosome spreads. This technique uses short polynucleotides of 500 or 600 bases; however, 2,000-4,000 bp polynucleotides are preferred. For a report on this technology, see Verma et al., Human Chromosome: Basic Technology Manual, Pergamon Press, New York (1
988).

For chromosome mapping, the polynucleotides can be individually (to label one chromosome or a site on that chromosome) or in multiples (to label multiple sites and / or multiple chromosomes). Can be used. The preferred polynucleotide corresponds to the non-coding region of the cDNA, as the coding sequence is believed to be more conserved within the gene family, thus increasing the likelihood of cross-hybridization during chromosome mapping. is there.

Once the polynucleotide has been mapped to a precise chromosomal location, the physical location of the polynucleotide can be used for linkage analysis. Linkage analysis can establish co-inheritance between chromosomal location and presentation of a particular disease. Disease mapping data is
For example, V. McKusick, “Mendelian Inheritance in Humans
Man) ”(available online from Johns Hopkins University Welch Medical Library). Assuming one megabase mapping analysis and one gene for every 20 kb, a cDNA precisely located in a chromosomal region associated with disease. Is 50
-One of the potentially 500 causative genes.

Thus, once co-inheritance is established, differences in polynucleotides and corresponding genes between affected and unaffected individuals can be identified. This analysis can be performed on individual humans using the polynucleotides of SEQ ID NOs: 1-19; 49-52; 57-72; and 107.

First, the macroscopically visible structural alterations of the chromosome, eg deletions or translocations, are examined in the chromosome spread or by PCR. If there are no structural changes, confirm the presence of point mutations. The mutation was observed in some or all of the affected individuals, but not in normal individuals, indicating that this mutation may induce disease. However, complete sequencing of polynucleotides and corresponding genes from several normal individuals requires the identification of mutations and polymorphisms. Once a new polymorphism is identified, this polymorphic polypeptide can be used for further linkage analysis.

Furthermore, increased or decreased expression of genes in affected individuals as compared to unaffected individuals can be assessed using the polynucleotides of the invention. Any of these changes (altered expression, chromosomal rearrangements, or mutations) can be used as a diagnostic or prognostic marker.

In addition to the above, polynucleotides can be used to control gene expression through the generation of triple-stranded helices or antisense DNA or RNA.
Both methods rely on the binding of polynucleotides to DNA or RNA. The preferred polynucleotides for these techniques are usually 20 to 40 bases in length, and are regions of genes involved in transcription (for a study of triple-stranded helix formation, see Lee et al., Nucl. Acids).
Res., 6: 3073 (1979); Cooney et al., Science, 241-456 (1988); and Dervan et al.
Science, 251: 1360 (1991)) or mRNA itself (for a review of antisense technology, Okano, J. Neurochem., 56: 560 (1991) and “Oligodeoxynucleotides (Oligodeoxynucleotides as antisense inhibitors of gene expression. -nucleot
ides as Antisense Inhibitors of Gene Expression) ”, CRC Press, Boca Rat
on, FL (1988)). Optimal DNA production for triple-stranded helix
And causes antisense RNA hybridization to inhibit the translation of the mRNA molecule into a polypeptide. Both techniques are effective in model systems and the information disclosed herein can be used to design antisense or triplex helix polynucleotides for treating diseases.

The polynucleotides of the present invention are also useful in gene therapy. One purpose of gene therapy is to insert a normal gene into an organism having a defective gene to correct the genetic abnormality. The polynucleotides disclosed in the present invention provide a highly accurate method for targeting such genetic abnormalities. Another purpose is to insert new genes that were not present in the host genome, thereby creating new properties in the host cell.

Polynucleotides are also useful in identifying individuals from small biological samples. For example, the US military is considering using restriction fragment length polymorphisms (RFLPs) to identify staff. In this technique, an individual's genomic DNA is digested with one or more kinds of restriction enzymes, and a Southern blotting is performed to obtain a band unique to the identification of staff. This method is used by the current "Dog T" to make reliable identification difficult, such as lost, switched or stolen.
It is not restricted by "ag (identification tag)". The polynucleotide of the present invention is RFL
Can be used as an additional DNA marker for P.

The polynucleotides of the present invention represent the actual base-by-base DN of selected portions of the individual's genome.
It can also be used as an alternative to RFLP by determining the A sequence. These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, and thus the sequence of the selected DNA can be determined. Because each individual has a unique set of DNA sequences, this technique makes it possible to identify the individual. If a unique ID database is built for each individual,
Individuals can be reliably identified, whether alive or dead, even from small tissue samples.

The use of DNA-based identification techniques as disclosed herein is also beneficial to forensic biology. DNA sequences collected from minute amounts of biological samples (eg, tissues such as hair and skin, body fluids such as blood, saliva, semen) can be amplified using PCR. In one of the prior art, polymorphic loci (e.g. DQa class II H
Individuals are identified using forensic biology using gene sequences amplified from (LA gene) (
Erlich, H., PCR Technology, Freeman and Co. (1992)). After amplifying these specific polymorphic loci, they were digested with one or more restriction enzymes, and Southern blot was performed using DNA corresponding to the DQa class H HLA gene as a probe to obtain a band set for identification. ing. Similarly, the polynucleotide of the present invention can be used as a polymorphic marker for forensic purposes.

There is also a need for reagents that can identify specific tissue sources. Such demands arise, for example, in the field of forensic medicine where an organization of unknown origin is brought about. Suitable reagents are, for example, those comprising DNA probes or primers specific for a particular tissue prepared from the sequences of the invention. Such a panel of reagents can identify tissues by species and / or species of organism. Similarly, these reagents can be used to screen tissue cultures for contamination.

[0124] At least, the polynucleotide of the present invention "excludes" a known sequence in the process of discovering a novel polynucleotide as a molecular weight marker in Southern gel, as a diagnostic probe for determining the presence or absence of specific mRNA in a specific cell type. As a probe to select and produce an oligomer to be attached to a "gene chip" or other support, to elicit anti-DNA antibodies using DNA immunization, and
It can be used as an antigen to induce an immune response.

Uses of Polypeptides The polypeptides identified in the present invention each have a wide range of uses. The following description is merely an example, and known techniques are used.

By using antibody-based techniques, the polypeptides of the present invention can be used to assay protein levels in biological samples. For example, classical immunohistological methods can be used to investigate protein expression in tissues (Jalkanen, M., et al., J. Cell. Biol., 101: 976-985 (1985); Jalkane.
n, M., et al., J. Cell. Biol., 105: 3087-3096 (1987)). As a method utilizing other antibodies useful for detecting protein gene expression, solid-phase enzyme immunoassay is available.
(ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels such as glucose oxidase, iodine ( ¹²⁵ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹¹² In) and technetium ( ^99m Tc), and other radioisotopes, fluorescent labels such as fluorescein and rhodamine, and biotin.

In addition to assaying the level of secreted protein contained in a biological sample, the protein can be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of proteins include those detectable by radiography, NMR or ESR. In the case of radiography, suitable labels include radioisotopes such as barium and cesium. While these radioisotopes emit detectable radiation, they do not cause any apparent harm to the subject. Markers suitable for NMR and ESR include those having a characteristic spin that can be detected (for example, deuterium), and they can be incorporated into an antibody by labeling the nutrients of the corresponding hybridoma.

Protein-specific prelabeled with a suitable detectable moiety such as a radioisotope (eg, ¹³¹ I, ¹¹² In, ^99m Tc), radiopaque material, or material detectable by nuclear magnetic resonance. The antibody or protein-specific antibody fragment is introduced into the mammal by, for example, the parenteral, subcutaneous or intraperitoneal route. It will be apparent in the art that the size of the subject and the imaging system used will dictate the amount of imaging moiety needed to produce a diagnostic image. In the case of radioisotope components, for human subjects, the amount of radioactivity injected is usually about 5-20 millicuries at ^99m Tc. The labeled antibody or labeled antibody fragment then preferentially accumulates at the location of cells which contain the particular protein. SW Burchiel et
al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragm
ents "describes in vivo tumor imaging (Tumor Imaging Chapter 13: Th
e Radiochemical Detection of Cancer, SW Burchiel and BA Rhodes, Ma
sson Publishing Inc. (1982)).

Thus, the present invention comprises (a) assaying the expression of a polypeptide of the present invention in cells or body fluids of an individual, (b) comparing the level of gene expression to a standard gene expression level, Disclosed is a method for diagnosing a disorder, which uses an increase / decrease in the gene expression level of a polypeptide to be assayed relative to a standard expression level as an indicator of the disorder.
Treatment of psychiatric disorders, including schizophrenia, and psychiatric disorders with neuroleptic drugs may be accompanied by dysregulation of neurotransmitter and / or neuropeptide levels, leading to upregulation or downregulation of polynucleotides or polypeptides. There is. Such changes can be diagnosed or observed by assaying for changes in polypeptide levels in tissues or body fluids such as CFS, blook or stool samples.

In addition, the polypeptides of the present invention can be used to treat diseases. For example, a patient may be administered a polypeptide of the invention to ameliorate the lack or reduced levels of the polypeptide (e.g. insulin) or the absence of another polypeptide (e.g. hemoglobin S in the case of hemoglobin B). And the decrease in levels,
For example, it inhibits the activity of an oncogene, activates the activity of a polypeptide (e.g., by binding to a receptor), or reacts with a free ligand (e.g., soluble TNF used to reduce inflammation). The activity of the membrane-bound receptor can be reduced or a desired response (eg, proliferation of blood vessels) can be generated by competing with the receptor.

Similarly, antibodies to the polypeptides of the present invention can be used to treat disease. For example, administration of an antibody to a polypeptide of the invention can stop and reduce overproduction of the polypeptide. Similarly, administration of an antibody can activate the polypeptide via binding to the polypeptide bound to the membrane (receptor) or the like. The polypeptide can also be used as an antigen to induce an immune response.

Local production of neurotransmitters and neuropeptides multimodally modulates neural function. For example, in schizophrenia, excess neurotransmitter activity is thought to be the source of psychotic behaviour. Administration of antibodies to overproduced polypeptides can modulate neural responses in mental disorders such as schizophrenia.

At a minimum, the polypeptides of the present invention can be used as molecular weight markers in SDS-PAGE gels or molecular sieve gel filtration columns by using methods well known to those of skill in the art. Polypeptides can also be used to raise antibodies and such antibodies may be used to measure protein expression from recombinant cells as a method of assessing host cell transformation. In addition, the polypeptides of the invention can be used to test the following biological activities.

Biological Activity The polynucleotides and polypeptides of the present invention can be used in assays to test one or more biological activities. If these polynucleotides and polypeptides show activity in a particular assay, it is likely that these molecules are involved in the disease associated with the biological activity. Thus, the polynucleotides and polypeptides of the invention can be used to treat related disorders.

Nervous System Activity The polypeptide or polynucleotide of the present invention activates or inhibits the proliferation, differentiation or migration (chemotactic) of neuroblasts, stem cells or glial cells, and thereby activates the central nervous system or the peripheral nervous system. It is believed to be useful in treating disorders or disorders of the nervous system. The polypeptide or polynucleotide of the present invention is a neuronal cell of a neurotransmitter, a neuromodulator and a mechanism of synaptic transmission, synthesis, metabolism and inactivation of a trophic factor, an enzyme, a structural protein, a membrane channel and a receptor neuron. By activating or inhibiting the mechanism of expression and integration in glial cells, or the mechanism of changes in the membrane composition of nerve cells, it is considered to be useful for the treatment of disorders or disorders of the central nervous system or peripheral nervous system.

The etiology of these deficiencies or disorders may be genetic, physical (eg, cancer or some autoimmune disorder), acquired (eg, due to chemotherapy or toxins), or caused by infection. To be Furthermore, the polynucleotides or polypeptides of the present invention can be used as markers or detectors for certain nervous system diseases or disorders. Such disorders or diseases include Alzheimer's disease, Pick's disease, Binswanger's disease, other senile dementia, Parkinson's disease, tremor palsy, obsessive-compulsive disorder, epilepsy, encephalopathy, ischemia, alcohol addiction, drug addiction, It may be any of schizophrenia, amyotrophic lateral sclerosis, multiple sclerosis, depression and bipolar manic depression. Alternatively, the polypeptides or polynucleotides of the invention can be used to investigate circadian changes, senescence, or long-term synergy, the latter acting on the hippocampus. Furthermore, particularly for mRNA species that occur in specific structures within the central nervous system, the polypeptide or polynucleotide of the present invention is used to perform complex behaviors such as learning and memory, emotions, drug addiction, glutamate neurotoxicity, and ingestion. Brain regions known to be involved in eating behavior, olfaction, viral infections, vision, and movement disorders can be investigated.

Immune Activity The polypeptides or polynucleotides of the invention are useful in the treatment of immune system deficiencies or disorders by activating or inhibiting the proliferation, differentiation or migration (chemotactic) of immune cells. Conceivable. Immune cells are generated through a process called hematopoiesis, and myeloid cells (platelets, erythrocytes, neutrophils, and macrophages) and lymphoid cells (B and T lymphocytes) are produced from pluripotent stem cells. . The etiology of these immune deficiencies or disorders may be genetic, physical (eg, cancer or some autoimmune disorder), acquired (eg, due to chemotherapy or toxins), or caused by infection. Further, the polynucleotide or polypeptide of the present invention is
It can be used as a marker or detector for a particular immune system disease or disorder.

The polynucleotides or polypeptides of the invention are believed to be useful in the treatment or detection of hematopoietic cell deficiencies or disorders. The polypeptides or polynucleotides of the invention can be used to enhance the differentiation and proliferation of hematopoietic cells, including pluripotent stem cells, to treat disorders involving the decline of certain (or many) hematopoietic cells. . Examples of immunodeficiency syndromes include blood protein disorders (e.g., agammaglobulinemia, hypogammaglobulinemia), ataxia-telangiectasia, unclassified immunodeficiency, Di George syndrome, HIV infection, HTLV. -BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia, phagocytic bactericidal dysfunction, severe combined immunodeficiency (SCI
Ds), Viscott-Aldrich disorder, anemia, thrombocytopenia, or hemoglobinuria, but is not limited thereto.

In addition, using the polypeptides or polynucleotides of the invention, hemostatic activity
It is also possible to modulate (bleeding inhibition) or thrombolytic activity (clot formation). For example, by improving the hemostatic activity or thrombolytic activity, using the polynucleotide or polypeptide of the present invention, blood coagulation disorders (e.g., fibrinogenemia, coagulation factor deficiency), platelet disorders (e.g., platelets). , Or wounds due to trauma, surgery or other causes. Alternatively, a polynucleotide or polypeptide of the invention that can reduce hemostatic or thrombolytic activity can be used to prevent or lyse coagulation. Such a molecule can cause a heart attack (
Infarction), stroke, or scarring.

The polynucleotides or polypeptides of the invention may also be useful in treating or detecting autoimmune disorders. Many autoimmune disorders result from inappropriate recognition of self as a non-self substance by immune cells. This improper recognition results in an immune response that results in the destruction of host tissue. Therefore, immune response, especially T cell proliferation,
Administration of the polynucleotides and polypeptides of the invention that inhibit differentiation, or chemotaxis, or in some way lead to the induction of tolerance, can be an effective treatment to prevent autoimmune disorders.

Examples of autoimmune disorders that can be treated or detected by the present invention include, but are not limited to, Addison's disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis. , Glomerulonephritis, Goodpasture syndrome, Graves' disease, multiple sclerosis, myasthenia gravis, neuritis, conjunctivitis, bullous pemphigoid,
Pemphigus, polyglandular endocrine disorder, purpura, Reiter's disease, Stiffman syndrome, autoimmune thyroiditis, systemic lupus erythematosus, autoimmune pneumonia, Guillain-Barre syndrome, insulin-dependent diabetes mellitus, and autoimmune inflammation Sexual eye disease. Schizophrenia has several aspects that suggest the involvement of autoimmune components in the process of this disease. Schizophrenic patients show immunological abnormalities, including hypersecretion of cytokines, the presence of antinuclear, anticytoplasmic and antiphospholipid antibodies, and a reduced CD4 + / CD8 + cell ratio.

Similarly, allergic reactions and symptoms, such as asthma (particularly allergic asthma) or respiratory disorders, can also be treated with the polypeptides or polynucleotides of the invention. In addition, these molecules can be used to treat anaphylaxis, hypersensitivity to antigenic molecules, or blood group mismatches.

The polynucleotides or polypeptides of the present invention can also be used for the treatment and / or prevention of organ rejection or graft-versus-host disease (GVHD). Organ rejection occurs when host immune cells destroy the transplanted tissue by an immune reaction. Similarly, GVHD also involves an immune response, in which non-self transplanted immune cells destroy host tissues. Administration of a polypeptide or polynucleotide of the present invention that inhibits an immune response, particularly T cell proliferation, differentiation, or chemotaxis, can be an effective treatment to prevent organ rejection or GVHD.

Similarly, the polypeptides or polynucleotides of the invention can also be used to reduce inflammation. For example, the polypeptide or polynucleotide can inhibit proliferation and differentiation of cells involved in an inflammatory response. These molecules are associated with infection-associated inflammation (eg, septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemic reperfusion injury, endotoxin mortality, arthritis, hyperacute rejection involving complement. , Both chronic and acute of inflammatory conditions, including nephritis, lung disorders caused by cytokines or chemokines, inflammatory bowel disease, Crohn's disease, or diseases resulting from overproduction of cytokines (eg, TNG or IL-1). It can be used to treat symptoms.

Hyperproliferative disorder polypeptides or polynucleotides can be used to treat or detect hyperproliferative disorders, including neoplasms. A polypeptide or polynucleotide of the invention can be directly or indirectly involved to inhibit the growth of that disorder.
Alternatively, the polypeptides or polynucleotides of the present invention are capable of growing other cells capable of inhibiting hyperproliferative disorders.

Hyperproliferative disorders can be treated, for example, by increasing the antigenicity of the immune response, particularly hyperproliferative disorders, or by T cell proliferation, differentiation or mobilization. This immune response can be increased either by enhancing the currently occurring immune response or by initiating a new immune response. Alternatively, reducing the immune response may also be a treatment for hyperproliferative disorders, such as treatment with chemotherapeutic agents.

Examples of hyperproliferative disorders that can be treated or detected by the polynucleotides or polypeptides of the present invention include, but are not limited to, neoplasms localized to the following organs: abdomen, bone, chest, digestive system. System, liver, pancreas, peritoneum, endocrine glands (adrenal gland, parathyroid gland, pituitary gland, testis, ovary, thymus, thyroid), eyes, head and neck, nerves (central and peripheral), lymphatic system, pelvic area, Skin, soft tissue, spleen, rib cage, and genitourinary system.

Similarly, other hyperproliferative disorders can also be treated or detected with the polynucleotides or polypeptides of the invention. Examples of such hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders,
Paraproteinemia, purpura, sarcoidosis, Sezary syndrome, Waardenstron macroglobulinemia, Gaucher disease, histiocytosis, and any other hyperproliferative disease localized to the previously mentioned organ system, as well as Neoplasm.

Infectious Diseases The polypeptides or polynucleotides of the invention can be used to treat or detect infectious agents. Infectious diseases can be treated, for example, by increasing the immune response, especially by increasing the proliferation and differentiation of B and / or T cells. This immune response can be increased either by enhancing the currently occurring immune response or by initiating a new immune response. Alternatively, the polypeptides or polynucleotides of the invention can also directly inhibit infectious agents without necessarily causing an immune response. In the case of schizophrenia, the infectious agent may contribute to the pathology, and the treatment of patients with the polypeptide or polynucleotide of the present invention acts as a vaccine that triggers a more effective immune response,
It is thought that it may change the progression of the disease.

Viruses are an example of infectious agents that can cause diseases or syndromes that can be treated or detected by the polynucleotides or polypeptides of the invention. Examples of viruses include, but are not limited to, the following DNA and RNA virus groups: Arbovirus, Adenoviridae, Arenaviridae, Arteritis virus, Bimaviridae, Bunyaviridae, Caliciviridae, Circo. Viridae, Coronaviridae, Flaviviridae, Hepadnaviridae (hepatitis), Herpesviridae (for example, cytomegalovirus, herpes simplex, herpes zoster), Mononega virus (for example, Paramyxoviridae, Measles virus, rapdoviridae, orthomyxoviridae (eg influenza)
, Papovaviridae, parvoviridae, picornaviridae, poxviridae (eg smallpox or complete smallpox), reoviridae (eg rotavirus), retroviridae (HTLV-I, HTLV-II, lentivirus) and Togaviridae (eg Rubella virus). The viruses included in these groups are
A variety of diseases or syndromes can be caused, including but not limited to:
Arthritis, bronchiolitis, encephalitis, eye infection (eg, conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E type, chronic active, delta type), meningitis, opportunistic infection ( AIDS), pneumonia, Burkitt lymphoma, chicken pox, hemorrhagic fever, measles, mumps, parainfluenza, rabies, common cold, polio, leukemia, rubella, sexually transmitted diseases, skin diseases (eg Kaposi's sarcoma, warts). And viremia. The polypeptides or polynucleotides of the invention can be used to treat or detect any of these syndromes or diseases.

Bacteria or fungi that can also cause diseases or syndromes and that can be treated or detected by the polynucleotides or polypeptides of the invention include, but are not limited to, the following Gram-negative and Gram-positive bacterial groups and fungi. Not: Actinomycetes (eg Corynebacterium, Mycobacterium, Nocardia),
Aspergillus, Bacillus (eg, B. anthracis, Clostridium), Bacteroidea, Blastomyces, Bordetella, Borrelia, Brucellosis, Candidiasis, Campylobacter, Coccidioithemycosis, Cryptococcosis, Dermatocycoses, Enteric Bacterium, (Klebsiella, Salmonella, Serratia, Erginia), Eridiveroslix, Helicobacter, Regenera, Leptospirosis, Listeria, Mycoplasma, Neisseria (eg Acinetobacter, gonorrhea, meningitis) Fungi), Pasteurella infectious diseases (eg Actinobacillus, Hemophilus, Pasteurella), Pseudomonas, Rickettsia, Chlamydiae, syphilis, and Staphylococcus. These bacteria or cardiomycotic groups can cause diseases or syndromes including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, Opportunistic infections (eg AIDS related infections), paronychia, prosthesis related infections, Reiter's disease, respiratory tract infections such as whooping cough or pyometra, sepsis, Lyme disease, cat scratch disease, dysentery, paratyphoid, food poisoning. , Typhus, pneumonia, gonorrhea, meningitis, chlamydia,
Syphilis, diphtheria, leprosy, paratuberculosis, tuberculosis, lupus, botulinum poisoning, gangrene, tetanus, impetigo, rheumatic fever, scarlet fever, sexually transmitted diseases, skin diseases (eg cellulitis, dermatocycoses), venomemia, urea infection. , Wound infections. The polypeptides or polynucleotides of the invention can be used to treat or detect any of these syndromes or diseases.

Further parasites responsible for diseases or syndromes that can be treated or detected with the polynucleotides or polypeptides of the invention include, but are not limited to, the following groups: amebiasis, babesiosis, coccidiosis, chestnut disease. Ptosporidiosis, binuclear amoebiasis, copulation, infestation, giardiasis, helminthosis, leishmaniasis, theileriosis, toxoplasmosis, trypanosomiasis and trichomoniasis. These parasites can cause a variety of diseases and syndromes including, but not limited to, scabies, scrub typhus, eye infections, intestinal diseases (eg, dysentery, giardia giardiasis), liver diseases, lung diseases. , Opportunistic infections (eg, AIDS-related), malaria, pregnancy complications, and toxoplasmosis. The polypeptides or polynucleotides of the invention can be used to treat or detect any of these syndromes or diseases.

Preferably treatment with a polypeptide or polynucleotide of the invention is
A method of administering an effective amount of a polypeptide to a patient, or removing cells from a patient, injecting the polynucleotide of the present invention into the cells, and returning the treated cells to the patient (ex v
ivo therapy) method. Furthermore, the polypeptides or polynucleotides of the invention can be used as antigens in vaccines to enhance the immune response to infectious diseases.

Regeneration The polypeptides or polynucleotides of the present invention can be used to differentiate, proliferate, attract cells and lead to tissue regeneration (Science, 276: 59-87 (199).
See 7)). Tissue regeneration can be congenital defects, trauma (wounds, burns, cuts or ulcers),
Tissues damaged by aging, disease (eg osteoporosis, osteoarthritis, periodontal disease, liver failure), surgery, fibrosis, reperfusion injury, or systemic cytokine damage, including plastic surgery Can be used to repair, replace, or protect.

Tissues that may be regenerated using the present invention include organs (eg pancreas, liver, small intestine, kidney, skin, endothelium), muscles (smooth muscle, skeletal muscle or myocardium), blood vessels (including vascular endothelium). , Nerves, hematopoiesis, and skeletal (bone, cartilage, tendons and ligaments) tissue. Regeneration is preferably carried out without traces or with only traces. Regeneration can also include angiogenesis.

Moreover, the polynucleotides or polypeptides of the present invention can enhance the regeneration of difficult-to-heal tissues. For example, increased tendon / ligament regeneration could reduce recovery time after injury. The polynucleotide or polypeptide of the present invention also comprises
It can also be used prophylactically due to its effect of avoiding damage. Specific diseases that can be treated include tendinitis, carpal tunnel syndrome, and other tendon or ligament defects.

Similarly, nerve and brain tissue can also be regenerated by proliferating and differentiating nerve cells using the polynucleotide or polypeptide of the present invention. Diseases that could be treated using this method include central and peripheral nervous system diseases, neuropathy, or physical and traumatic disorders (eg myelopathy, head injury, cerebrovascular disease, and stroke). In particular, diseases associated with peripheral neuropathy, peripheral neuropathy (eg due to chemotherapy or other medical therapies), local neuropathy, and central nervous system diseases (eg Alzheimer's disease, Parkinson's disease, Huntington's disease, muscular atrophy) It is believed that lateral sclerosis, and Shy-Drager syndrome) can all be treated with the polynucleotides or polypeptides of the invention.

Chemotaxis A polynucleotide or polypeptide of the invention can have chemotactic activity. Molecules with chemotactic properties are found in cells (eg monocytes, fibroblasts, neutrophils, T cells,
Mast cells, eosinophils, epithelial cells and / or endothelial cells) are attracted or recruited to specific sites in the body, such as sites of inflammation, infection or hyperproliferation. The mobilized cells can subsequently overcome and / or heal certain traumas or abnormalities.

The polynucleotides or polypeptides of the present invention can enhance the chemotactic activity of particular cells. These chemotactic molecules then increase the number of cells that act on specific local targets in the body, resulting in inflammation, infection, hyperproliferative disorders,
Or it can be used to treat any immune system disorder. For example, chemotactic molecules can be used in the treatment of tissue wounds and other trauma by attracting immune cells to the damaged local area. The chemotactic molecule of the present invention can also attract fibroblasts and thus can be used in the treatment of wounds.

It is also possible that the polynucleotides or polypeptides of the invention may be capable of inhibiting chemotactic activity. These molecules could also be used to treat disorders. Thus, it is considered that the polynucleotide or polypeptide of the present invention can be used as a chemotaxis inhibitor.

Binding Activity A polypeptide or polynucleotide of the invention can be used to screen for molecules that bind to this polypeptide, or to which this peptide binds. The binding of a molecule to a polypeptide can activate (ie, agonist), increase, inhibit (ie, antagonist), or decrease the activity of the bound polypeptide or molecule. Examples of such molecules include antibodies, oligonucleotides, proteins (eg receptors), or low molecular weight molecules.

Preferably, the molecule is the natural ligand of the polypeptide, eg, a fragment of the ligand, or a natural substrate, ligand, structurally or functionally similar substance (Coli).
gan et al. Current Protocols in Immunology, 1 (2), Chpter 5 (1991))
Closely related to. Similarly, the molecule may be intimately associated with the natural receptor to which the polypeptide binds, or at least to a fragment of the receptor capable of binding the polypeptide (eg, the active site). In either case, the molecule can be rationally designed by known techniques.

Preferably screening for these molecules involves the production of suitable cells which express the polypeptide as a secreted protein or on the cell membrane. Preferred cells include cells of mammalian, yeast, Drosophila or E. coli origin. Next, preferably, a cell expressing the polypeptide (or a cell membrane containing the expressed polypeptide) is contacted with a compound to be measured, which potentially contains this molecule, and binding,
Examine the activation, or inhibition, of the activity of either the polypeptide or the molecule.

The assay can simply examine binding of a candidate compound to the polypeptide, where binding is detected by the label or in an assay involving competition with a labeled competitor. In addition, the assay can test whether the candidate compound gave a signal that results from binding to the polypeptide.

Alternatively, the assay can be performed in cell-free conditions, with solid phase support-immobilized polypeptides / molecules, chemical libraries, or mixtures of natural products. The assay may also simply include the step of mixing the compound to be measured with a solution containing the polypeptide, measuring the activity or binding of the polypeptide / molecule, and comparing the activity or binding of the polypeptide / molecule to a standard. it can.

[0166] Preferably, the ELISA assay uses a monoclonal antibody or a polyclonal antibody to measure the polypeptide level or activity in a sample (eg, biological sample). The antibody can directly or indirectly bind to the polypeptide or compete with the polypeptide for a substrate to measure the level or activity of the polypeptide.

All of these above assays can be used as diagnostic or prognostic markers. Molecules found using these assays can be used to activate or inhibit polypeptides / molecules, to treat a disease or to bring a particular result to a patient (vascular growth). it can. In addition, these assays can find substances that can inhibit or increase the production of the polypeptide from appropriately treated cells or tissues.

The invention thus includes a method of identifying a compound that binds to a polypeptide of the invention, including the steps of: (a) incubating a candidate binding compound with a polypeptide of the invention; Then, (b) it is measured whether or not they are bound. The invention further includes methods of identifying agonists / antagonists, including the steps of: (a) incubating a candidate compound with a polypeptide of the invention, (b) assaying biological activity, and ( c) Determine if the biological activity of the polypeptide is altered.

Other Activities The polypeptides or polynucleotides of the invention can also increase or decrease embryonic stem cell differentiation or proliferation, as well as the hematopoietic chain as described above.

The polypeptides or polynucleotides of the invention may also include height, weight, hair color,
It can be used to regulate mammalian characteristics such as eye color, skin, adipocyte percentage, pigmentation, size, and shape (eg, cosmetic surgery). Similarly,
The polypeptides or polynucleotides of the invention can be used to regulate mammalian metabolism that affects catabolism, anabolic activity, processing, utilization, and energy storage.

Polypeptides or polynucleotides of the invention may be used in biorhythms, circadian rhythms, depressive states (including depressive disorders), propensity to violence, resistance to opiates and opioids, tolerability of opiates and opioids, opiates. And opioid withdrawal, fertility (preferably due to activin or inhibin-like activity), hormonal or endocrine levels, appetite, sexual drive,
It can be used to alter a mammal's mental or physical state by affecting memory, stress, or other cognitive levels.

The polypeptides or polynucleotides of the invention may also have a storage capacity, fat content,
It can be used as a food additive or preservative, such as to increase or decrease lipids, proteins, carbohydrates, vitamins, minerals, cofactors, or other nutritional ingredients.

Other Preferred Embodiments Where the polynucleotide of the invention is downregulated and aggravates a pathological condition such as psychosis or other neuropsychiatric disorder, treatment, prevention, amelioration of such pathological condition or To effect the regulation, the expression of the polynucleotide can be increased or the level of the intact polypeptide product can be increased. This can be achieved, for example, by administering a polynucleotide or polypeptide of the invention to a mammalian subject.

The polynucleotide of the present invention can be administered to a mammalian subject by a recombinant expression vector containing the polynucleotide of the present invention. The mammalian subject is a human,
Baboon, chimpanzee, macaque, cow, horse, sheep, pig, horse, dog, cat,
It can be a rabbit, guinea pig, rat, or mouse. Preferably, the recombinant vector is the polynucleotide set forth in SEQ ID NOS: 1-19; SEQ ID NOS: 49-52; SEQ ID NOS: 57-72 and SEQ ID NO: 107, or SEQ ID NOS: 1-19; SEQ ID NOS: 49-52; 57-72 and polynucleotides having at least 98% identity to the nucleic acid sequences set forth in SEQ ID NO: 107. Also, preferably, the recombinant vector is SEQ ID NO: 1-19; SEQ ID NO: 49-52; SEQ ID NO: 5
7 to 72 and SEQ ID NO: 107 are included in the variant polynucleotide having at least 80%, 90%, or 95% identity.

Administration of a polynucleotide or recombinant expression vector of the invention to a mammalian subject is used to express the polynucleotide in said subject, for example to treat psychosis or other neuropsychiatric disorders. You can Expression of the polynucleotide in target cells, including but not limited to striatal and nucleus accumbens cells, results in greater production of the encoded polypeptide. In some cases where the encoded polypeptide is a nuclear protein, regulation of other genes may be upregulated or downregulated secondarily.

As described above, one of skill in the art can utilize a number of suitable viral and non-viral methods for introducing nucleic acid molecules into target cells. In addition, naked polynucleotides can be administered to target cells. The polynucleotide and recombinant expression vector of the present invention can be administered as a pharmaceutical composition. Such compositions include an effective amount of the polynucleotide or recombinant expression vector and a pharmaceutically acceptable combination selected for compatibility with the mode of administration. Suitable compounding materials are preferably non-toxic to the recipients at the concentrations used and, for example, pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, The dissolution or release rate, adsorption, or penetration of the composition can be modified, maintained, or maintained. Remington's P
Pharmaceutical Sciences (18th Edition, AR Genn
aro, Mack Publishing Company, 1990).

The pharmaceutically active compounds (ie, polynucleotides or vectors) can be processed according to conventional pharmaceutical methods to produce a medicament that is administered to patients, including humans and other mammals. Accordingly, pharmaceutical compositions containing polynucleotides or recombinant expression vectors can be made in solid form (including granules, powders, or suppositories) or liquid form (eg, solutions, suspensions, or emulsions). it can.

Dosage regimens for treating disease with compositions comprising polynucleotides or expression vectors include the type or severity of psychosis or other neuropsychiatric disorder, the age, weight, sex, medical condition, administration of the patient. Based on various factors including the route and the particular compound used. Thus, the dosing regimen can vary widely, but can be routinely determined using standard methods. A typical dosage may range from about 0.1 mg / kg to about 100 mg / kg or more, depending on the factors mentioned above.

The frequency of dosing will depend on the pharmacokinetic parameters of the polynucleotide or vector in the formulation being used. Typically, the physician will administer the composition until a dosage is reached that achieves the desired effect. Thus, the composition may comprise two or more administrations over time as a single administration, which may contain the same amount of the desired molecule,
Or not necessarily in the same amount) or as a continuous infusion with an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ordinary skill of those in the art. Appropriate doses can be ascertained by use of appropriate dose response data.

Cells of a mammalian subject can be in vivo, ex vivo or in vitro.
It can be transfected with ro. Administration of a polynucleotide or a recombinant vector containing a polynucleotide to a target cell in vivo can be accomplished using any of a variety of techniques well known to those of skill in the art.
For example, US Pat. No. 5,672,344 discloses recombinant neurotropic HSV-1.
A virus-mediated gene transfer system in vivo with vectors has been described. The above compositions of polynucleotides and recombinant vectors can be transfected in vivo by oral, buccal, parenteral, rectal, or topical administration, as well as by inhalation spray. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intrasternal, or intraperitoneal techniques.

Although the nucleic acids and / or vectors of the invention may be administered as the sole active pharmaceutical agent, they may also be used in combination with one or more vectors of the invention or other agents. You can When administered as a mixture,
The therapeutic agents can be formulated as separate compositions that are administered at the same time or at different times. Alternatively, the therapeutic agents can be administered as a single composition.

Another delivery system for the polynucleotides of the present invention is a "non-viral" delivery system. The techniques used or proposed for gene therapy include DNA
- ligand complexes, adenovirus -. Ligand -DNA complexes, direct DNA injection, CaPO ₄ precipitation, gene gun techniques, electroporation, include lipofection, and colloidal dispersions (Mulligan, R, (1993
) Science, 260 (5110): 926-32 (1993)). Any of these methods are widely available to those skilled in the art and are suitable for use in the present invention. It should be understood that other suitable methods are available to those of skill in the art, and the present invention can be accomplished using any of the available methods of transfection. Several such methods have been used by those of skill in the art with varying success (Mulligan, R., (1993) Sci.
ence, 260 (5110): 926-32 (1993)).

Where the polynucleotides of the invention are upregulated and aggravate a pathological condition (such as psychosis or other neuropsychiatric disorder) in a mammalian subject, treatment, prevention, amelioration of such pathological condition, Alternatively, the expression of the polynucleotide can be blocked or reduced, or the level of the intact polypeptide product can be reduced to effect the regulation. This is, for example, antisense
This can be achieved by the use of oligonucleotides or ribozymes. Alternatively, a drug or antibody that binds to and inactivates the polypeptide product can be used.

Antisense oligonucleotides are nucleotide sequences that are complementary to a particular DNA or RNA sequence. When a complementary nucleotide is introduced into a cell, the complementary nucleotide binds with the native sequence produced by the cell to form a complex, blocking either transcription or translation. Preferably, the antisense oligonucleotide is at least 11 nucleotides in length,
It can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides in length. Longer sequences can also be used. Antisense oligonucleotide molecules can be provided in DNA constructs and introduced into cells as described above to reduce the level of the gene product of the invention within the cell.

Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides include alkyl phosphonates, phosphorothioates, phosphorodithioates, alkyl phosphonothioates, alkyl phosphonates, phosphoramidates, phosphates, carbamates, acetamates, carboxymethyl esters, carbonates, and phosphates. A non-phosphodiester internucleotide linkage, such as a fertotriester, is used to covalently link the 5'end of a nucleotide to the 3'end of another nucleotide, either manually or by an automated synthesizer. Can be synthesized by. Brown, (1994) Meth. Mol. Biol. , 20
: 1-8; Sonveaux, (1994) Meth. Mol. Biol. Two
6: 1-72; Uhlmann et al., (1990) Chem. Rev. , 90: 5
43-583.

Altered gene expression can be obtained by designing antisense oligonucleotides that form a duplex with the control, 5 ′ or regulatory regions of the genes of the invention. Transcription initiation site (eg, -10 and +10 from the initiation site)
Oligonucleotides derived from the site between the positions) are preferred. Similarly, inhibition
This can be accomplished using a "triple helix" base pairing methodology. Triple helix pairing is useful to prevent the double helix from opening sufficiently for the binding of polymerases, transcription factors, or chaperones. Therapeutic advances using triplex DNA have been described in the literature (eg Gee et al., MOLECULAR).
AND IMMUNOLOGIC APPROACHES (Huber &
Carr), Futur Publishing Co. , Mt. Kisco
, N .; Y. , 1994). Antisense oligonucleotides can also be designed to block translation of mRNA by preventing the transcript from binding to the ribosome.

Exact complementarity is not required for successful formation of a complex of antisense oligonucleotides with the complementary sequence of a polynucleotide. For example, containing two, three, four or more regions of contiguous nucleotides that are exactly complementary to a polynucleotide, each of which is a contiguous nucleotide that is not complementary to an adjacent nucleotide. Antisense oligonucleotides separated by regions may provide sufficient targeting specificity for mRNA. Preferably, each region of complementary contiguous nucleotides is at least 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides or more in length. Non-complementary intervening sequences preferably have a length of 1
Nucleotides, 2 nucleotides, 3 nucleotides, or 4 nucleotides. One of skill in the art can readily use the calculated melting point of an antisense-sense pair to determine the degree of mismatch that is tolerated between a particular antisense oligonucleotide and a particular polynucleotide sequence. .

Antisense oligonucleotides can be modified without affecting their ability to hybridize to the polynucleotides of the invention. These modifications can be internal to the antisense molecule or can be present at one or both ends of the antisense molecule. For example, the phosphate bond between the nucleotides can be modified by adding a cholesteryl or diamine component while varying the number of carbon residues between the amino group and the terminal ribose. Modified bases and / or sugars (such as arabinose in place of ribose, or 3 ', 5'-substituted oligonucleotides in which the 3'hydroxyl or 5'phosphate groups are substituted) are also modified anti- It can be used in sense oligonucleotides. These modified oligonucleotides can be prepared by methods well known in the art. For example, Ag
Rawal et al., (1992) Trends Biotechnol. 10: 1
52-158; Uhlmann et al., (1990) Chem. Rev. , 90: 5
43-584; Uhlmann et al., (1987) Tetrahedron. Le
tt. 215: 3539-3542.

Ribozymes are RNA molecules with catalytic activity. For example, Cech, (1
987) Science, 236: 1532-1539; Cech, (1990).
) Ann. Rev. Biochem. , 59: 543-568; Cech, (1
992) Curr. Opin. Struct. Biol. 2: 605-609
Couture & Stinchcomb, (1996) Trends G
enet. , 12: 510-515. Ribozymes can be used to inhibit gene function by cleaving RNA sequences, as is known in the art (eg Haseloff et al., US Pat. No. 5,64).
1, 673). The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.

The coding sequence of a polynucleotide of the invention can be used to make a ribozyme that specifically binds to the mRNA transcribed from the polynucleotide. Methods have been developed and described in the art for designing and constructing ribozymes capable of cleaving RNA molecules in trans in a highly sequence-specific manner (Has
Eloff et al. (1988) Nature, 334: 585-591). For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering distinct "hybridization" regions into the ribozyme. The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (eg Ge.
rrach et al., EP 321,201).

Specific ribozyme cleavage sites within an RNA target can be identified by scanning the target molecule for ribozyme cleavage sites that include the sequences below. That is, GUA, GUU, and GUC. A short RN between 15 and 20 ribonucleotides, corresponding to the region of the target RNA containing the cleavage site
Once the A sequence is identified, its RNA sequence can be evaluated for secondary structural features that can render the target inoperable. The suitability of candidate RNA targets can also be evaluated by examining their accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. The nucleotide sequences set forth in SEQ ID NOs: 1-19; SEQ ID NOs: 49-52; SEQ ID NOs: 57-72 and SEQ ID NO: 107, and their complements, provide a suitable source of hybridization region sequences. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for its target. The hybridization and cleavage regions of the ribozyme can be linked together such that the catalytic region of the ribozyme can cleave the target when hybridized to the target RNA via the complementary region.

Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation can be used to introduce the ribozyme-containing DNA construct into cells where it is desired to reduce expression of the polynucleotide. Alternatively, if the cell is desired to stably retain such a DNA construct, can the construct be supplied in a plasmid and maintained as a separate element, as is known in the art, Alternatively, it can be integrated into the genome of the cell. The DNA construct encoding the ribozyme can include transcriptional regulatory elements such as promoter elements, enhancers or UAS elements, and transcription termination signals to control transcription of the ribosome in the cell.

Ribozymes can be engineered to produce ribozyme expression in response to factors that induce expression of the target gene, as taught by Haseloff et al., US Pat. No. 5,641,673. . Ribozymes can also be engineered to provide additional levels of regulation, resulting in mRNA destruction only when both the ribozyme and the target gene are introduced intracellularly.

Manufacture of Diagnostic Tests The methods of the invention can be used to diagnose pathological conditions such as psychosis or other neuropsychiatric disorders or susceptibility to such pathological conditions. Testing for the expression of the polynucleotides of the invention or the presence of polynucleotide products can give results that correlate with the severity of the pathological condition and can direct appropriate treatment. For example, the presence or absence of a mutation in the polynucleotide of the present invention can be determined, and based on the presence or absence of this mutation, a pathological condition or susceptibility to a pathological condition can be diagnosed. Furthermore, a change in the expression of the polypeptide encoded by the polynucleotide of the present invention can be detected, and if there is a change in the expression of the polypeptide, this is a pathological condition or a pathological condition. It is an index of susceptibility. The change in expression may be an increase in expression level or a decrease in expression level.

As a further diagnostic method of the present invention, a first biological sample may be obtained from a patient suspected of having a pathological condition such as psychosis or addiction-related behavior, with which an appropriate comparison is made. A second sample is obtained from the resulting control source. Biological samples are saliva,
It can include tissues such as blood, cerebrospinal fluid, amniotic fluid, urine, feces, or gastrointestinal tissue. Suitable control sources can be obtained from one or more mammalian subjects that do not have a pathological condition. For example, the average concentration and distribution of a polynucleotide or polypeptide of the invention may be determined from biological populations obtained from a representative population of mammalian subjects, where the mammalian subject is the same species as the subject from which the test sample is taken. It can be determined from the sample. The amount of at least one polypeptide encoded by the polynucleotide of the invention is determined for the first and second samples. First
Comparing the amount of polypeptide in the second sample with that of the second sample. A patient is diagnosed as having a pathological condition if the amount of polypeptide in the first sample is within the range of samples obtained from a representative group of patients having a pathological condition.

Other preferred embodiments of the claimed invention are SEQ ID NOS: 1-19;
49-52; 57-72 and 107 nucleotide sequences that are at least 80%, preferably at least 85%, more preferably at least 90%, most preferably 95% identical to a sequence of at least about 50 contiguous nucleotides. It includes an isolated nucleic acid molecule that comprises a nucleotide sequence.

Further, the contiguous nucleotide sequences are represented by SEQ ID NOs: 1 to 19; 49 to 52;
Also preferred are nucleic acid molecules contained within the nucleotide sequence of 7-72 and 107 and located within a region starting with the nucleotide located near the 5'nucleotide of the clone sequence and ending with the nucleotide located near the 3'nucleotide of the clone sequence.

In addition, the contiguous nucleotide sequences are SEQ ID NOS: 1-19; 49-52;
Included in the contiguous nucleotide sequence from 7 to 72 and 107, 5 of the start codon
'Starting from a nucleotide located near the nucleotide, SEQ ID NOs: 1-19; 4
Also preferred are nucleic acid molecules that lie within a region terminating at a nucleotide located near the 3'nucleotide of the cloned sequences defined for 9-52; 57-72 and 107.

Similarly, the contiguous nucleotide sequences are SEQ ID NOs: 1-19; 49-52;
For nucleotides contained in the contiguous nucleotide sequence of 57-72 and 107 and located near the 5'nucleotide of the first amino acid of the signal peptide, for SEQ ID NOs: 1-19; 49-52; 57-72 and 107 Also preferred are nucleic acid molecules that lie within a region ending with a nucleotide located near the 3'nucleotide of the defined clone sequence.

Also, a sequence of at least about 150 consecutive nucleotides and at least 9 of the nucleotide sequences of SEQ ID NOs: 1-19; 49-52; 57-72 and 107.
Also preferred is an isolated nucleic acid molecule that comprises a nucleotide sequence that is 5% identical.

Further isolated is isolated comprising a nucleotide sequence that is at least 95% identical to a sequence of at least about 500 contiguous nucleotides in the nucleotide sequences of SEQ ID NOs: 1-19; 49-52; 57-72 and 107. Also preferred are nucleic acid molecules.

A further preferred embodiment is a nucleotide sequence that is at least 95% identical to SEQ ID NOs: 1-19; 49-52; 57-72 and 107, located near the 5'nucleotide of the first amino acid of the signal peptide. A nucleic acid molecule comprising a nucleotide sequence starting with the nucleotide sequence starting with the nucleotide sequence ending with the nucleotide sequence located near the 3'nucleotide of the clone sequence defined for SEQ ID NOs: 1-19; 49-52; 57-72 and 107.

A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to the complete nucleotide sequence of SEQ ID NOs: 1-19; 49-52; 57-72 and 107. In another embodiment, the invention features SEQ ID NO: 1.
-19; 49-52; 57-72 and 107, a method for detecting in a biological sample a nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to a complete nucleotide sequence selected from the group consisting of: Providing a method comprising comparing at least one nucleic acid molecule therein with a sequence selected from said group and determining whether the sequence of said nucleic acid molecule is at least 95% identical to said selected sequence.

An isolated nucleic acid molecule that hybridizes to a nucleic acid molecule under stringent hybridization conditions, wherein the hybridizing nucleic acid molecule has a nucleotide sequence consisting of only A residues or T residues. The nucleic acid molecule that it has is preferably a nucleic acid molecule that does not hybridize under stringent hybridization conditions.

A further preferred embodiment is a nucleotide that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-19; 49-52; 57-72 and 107. A method of detecting a nucleic acid molecule comprising a sequence in a biological sample, wherein at least one nucleic acid molecule in the sample is compared to a sequence selected from the group, wherein the sequence of the nucleic acid molecule is selected A method comprising determining whether the sequences are at least 95% identical.

[0206] Also, in the above method, wherein the step of comparing sequences comprises measuring the degree of hybridization between a nucleic acid molecule in the sample and a nucleic acid molecule comprising the sequence selected from the group. Is also preferable. Similarly, in the above method,
Also preferred is a method wherein the step of comparing sequences is carried out by comparing the nucleotide sequences determined from the nucleic acid molecules in the sample with the sequences selected from the group. Nucleic acid molecules can include DNA or RNA molecules.

Further, a preferred embodiment is a method of identifying the species, tissue or cell type of a biological sample, SEQ ID NOs: 1-19; 49-52; 57-72, if any.
And a nucleic acid molecule containing a nucleotide sequence at least 95% identical to a sequence of at least 50 contiguous nucleotides in the nucleotide sequence selected from the group consisting of 107 and 107 in the sample.

Also provided is a method of diagnosing a pathological condition associated with an abnormal structure or expression of a gene in a subject, comprising SEQ ID NOS: 1-19; 49-52; 57, if any.
At least 5 in the nucleotide sequence selected from the group consisting of
Also preferred is a method comprising detecting a nucleic acid molecule comprising a nucleotide sequence that is at least 95% identical to a sequence of 0 contiguous nucleotides in a biological sample obtained from said subject.

A method of diagnosing a pathological condition is provided in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 50 consecutive nucleotides of a sequence selected from said group. Of at least 95% identity to the sequence of SEQ ID NO. 1).

Also provided is a composition of matter comprising an isolated nucleic acid molecule, the nucleotide sequence of said nucleic acid molecule comprising a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is SEQ ID NO: Compositions that are at least 95% identical to a sequence of at least 50 contiguous nucleotides selected from the group consisting of 1-19; 49-52; 57-72 and 107 nucleotide sequences are also preferred. Nucleic acid molecules can include DNA or RNA molecules.

Also, a single amino acid sequence that is at least 90% identical to the sequence of at least about 10 consecutive amino acids in the amino acid sequence translated from SEQ ID NOs: 1-19; 49-52; 57-72 and 107. Separated polypeptides are also preferred.

Further, the above-mentioned sequences of consecutive amino acids are represented by SEQ ID NOs: 1 to 19; 49 to 52; 57.
Included in the acids in the amino acid sequence translated from ~ 72 and 107, starting from the residue located near the first amino acid of the secreted portion and located near the last amino acid of the open reading frame. Polypeptides within the range ending with residues are also preferred.

Also, a single amino acid sequence that is at least 95% identical to a sequence of at least about 30 consecutive amino acids in the amino acid sequence translated from SEQ ID NOs: 1-19; 49-52; 57-72 and 107. Separated polypeptides are also preferred.

In addition, a single amino acid sequence that is at least 95% identical to a sequence of at least about 100 consecutive amino acids in the amino acid sequence translated from SEQ ID NOs: 1-19; 49-52; 57-72 and 107. Separated polypeptides are also preferred.

Also preferred is an isolated polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acids in the amino acid sequences translated from SEQ ID NOs: 1-19; 49-52; 57-72 and 107.

Further, a peptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 consecutive amino acids in the amino acid sequence translated from SEQ ID NOs: 1-19; 49-52; 57-72 and 107. A method of detecting in a sample, the amino acid sequence of at least one polypeptide molecule in said sample being compared to a sequence selected from said group, wherein the sequence of polypeptide molecules in said sample is at least 10 Preferred is a method which comprises determining whether it is at least 90% identical to said contiguous sequence of amino acids.

Also, in the above method, the step of comparing the amino acid sequence of at least one polypeptide molecule in the sample with a sequence selected from the group is
19; 49-52; 57-72 and a sequence of at least 10 consecutive amino acids in a sequence selected from the group consisting of amino acid sequences translated from 107 and 90
Also preferred is a method comprising determining how much the polypeptide in the sample binds to an antibody that specifically binds to a polypeptide containing amino acid sequences that are% identical.

Also preferred is the above method, wherein said step of comparing sequences is carried out by comparing an amino acid sequence determined from a polypeptide molecule in said sample with said sequence selected from said group.

Also provided is a method of identifying a species, tissue or cell type in a biological sample, the method being selected from the group consisting of amino acid sequences translated from SEQ ID NOs: 1-19; 49-52; 57-72 and 107. Also preferred is a method comprising the step of detecting in a biological sample, if any, a polypeptide molecule comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in the sequence.

The above method for identifying a species, tissue or cell type of a biological sample also comprising detecting a polypeptide molecule comprising an amino acid sequence in a panel of at least two amino acid sequences, said method comprising: At least one sequence in the panel comprises at least 10 contiguous amino acid sequences selected from the above group and at least 9 sequences.
A method of 0% identity is also preferred.

Also provided is a method of diagnosing a pathological condition associated with an abnormal structure or expression of a gene in a subject, comprising a polypeptide molecule comprising an amino acid sequence in a panel of at least two amino acid sequences, At least one sequence in said panel is at least 90 with a sequence of at least 10 contiguous amino acids selected from the group consisting of amino acid sequences translated from SEQ ID NOs: 1-19; 49-52; 57-72 and 107. Also preferred is a method comprising the step of detecting, if any, polypeptide molecules that are% identical, in a biological sample obtained from said subject.

In any of these methods, the step of detecting the polypeptide molecule comprises using an antibody. In addition, an amino acid sequence at least 90% identical to a sequence of at least 10 consecutive amino acids in a sequence selected from the group consisting of amino acid sequences translated from SEQ ID NOs: 1 to 19; 49 to 52; 57 to 72 and 107 Also preferred is an isolated nucleic acid molecule that comprises a nucleotide sequence that is at least 95% identical to a nucleotide sequence that encodes a polypeptide that comprises.

Also preferred is an isolated nucleic acid molecule in which said nucleotide sequence encoding the polypeptide is optimized for expression of said polypeptide in a prokaryotic host.

Also, an isolated nucleic acid encoding a polypeptide whose nucleotide sequence comprises an amino acid sequence selected from the group consisting of amino acid sequences translated from SEQ ID NOs: 1-19; 49-52; 57-72 and 107. Molecules are also preferred.

Further preferred is a method of producing a recombinant vector comprising inserting any of the above isolated nucleic acid molecules into a vector. A recombinant vector produced by this method is also preferable. A method for producing a recombinant host cell by introducing a vector into the host cell together with the recombinant host cell produced by this method is also preferable.

Also preferred is a method of producing an isolated polypeptide which comprises culturing the recombinant host cell under conditions such that the polypeptide is expressed and recovered. Further, the recombinant host cell is a eukaryotic cell, and the polypeptide has SEQ ID NOs: 1 to 1.
9; 49-52; 57-72 and 107, which produces an isolated polypeptide comprising a secreted portion of a human secretory protein comprising an amino acid sequence selected from the group consisting of: The method is also preferred. Also preferred is an isolated polypeptide produced by this method.

Further, a method of treating an individual in need of increased levels of secretory protein activity, wherein said individual is provided with an isolated polypeptide, polynucleotide or antibody according to the claimed invention. Also preferred is a method of prescribing to such individuals a pharmaceutical composition comprising an amount effective to effectively increase the level of protein activity in said individuals.

The present invention also includes a diagnostic system, preferably in kit form, which is used in a body sample (brain tissue, cell suspension or tissue section, or body fluid sample such as CSF, blood, plasma or serum). The presence of the polypeptide of the present invention is assayed, and the presence, and preferably the amount, of the polypeptide of the present invention is desirably detected according to the diagnostic method described herein.

In a related embodiment, the gene corresponding to the mRNA that is diagnostic for the presence or expression of the polynucleotide of the invention or the polynucleotide of the invention or the polypeptide of the invention present in the cell in a cell. A nucleic acid molecule can be used as a probe (oligonucleotide) for detecting the presence of It will typically be from about 20 to 500 nucleotides in length, but various lengths from at least about 10 are suitable, with lengths from about 10 to about 5000 nucleotides being suitable. Hybridization methods are very well known to the person skilled in the art and will not be described further here.

In a related embodiment, the detection of the gene corresponding to the polynucleotide of the present invention can be carried out by a primer extension reaction such as polymerase chain reaction (PCR). For this purpose, PCR primers are used in pairs, which are well known, based on the nucleotide sequence of the gene to be detected. Preferably, the nucleotide sequence is part of the nucleotide sequence of the polynucleotide of the invention. Particularly preferred PCR primers can be derived from any part of the DNA sequence encoding the polypeptide of the invention, but are preferably from regions that are not conserved in other cellular proteins.

PCR primer pairs useful in detecting the genes corresponding to the polynucleotides of the invention and the expression of these genes are described in the examples (and corresponding table). Nucleotide primers from the corresponding regions of the polypeptides of the invention described herein are easily prepared and PCR for detection of the presence or expression of the corresponding gene in any of a variety of tissues. Used as a primer.

The diagnostic system comprises a separately packaged reagent of a subject polypeptide, subject antibody or monoclonal antibody of the invention, and / or a nucleic acid molecule probe of the invention of the invention in an amount sufficient for at least one diagnosis. Included in.

In another embodiment, the diagnostic system preferably comprises a polypeptide of the invention or an antibody immunoreactive with the polypeptide of the invention when administered for therapeutic purposes, preferably in the form of a kit. For the purpose of, for example, monitoring the change, it is possible to assay the presence of the polypeptide of the present invention or an antibody immunoreactive with the polypeptide of the present invention in a body fluid sample. The system comprises a polypeptide of the invention and / or a subject animal antibody as a separately packaged immunochemical reagent in an amount sufficient for at least one assay.

Instructions for use of the packaged reagents are also typically included. As used herein, a “package” is glass, plastic (eg, polyethylene, polypropylene or polycarbonate), paper, foil and the like, and is not limited to the polypeptides, polyclonal or monoclonal antibodies of the invention. Refers to a solid matrix or material that can be retained within a range. Thus, for example, if a milligram amount of the polypeptide or antibody is to be used, a glass vial can be used as the package, or microtiter plate wells can be used to manipulate the microgram amount of the polypeptide. Immobilized (ie, immunologically bound by an antibody or antigen, respectively).

“Instructions for use” typically refer to reagent concentration or at least one diagnostic method parameter (quantitative relationship of reagent and sample to be mixed, retention time of reagent / sample mixture, temperature, buffer conditions). , Etc.) as described above.

The diagnostic system of the present invention preferably further comprises a label or labeling means capable of indicating the formation of an immune complex comprising the polypeptide or antibody molecule of the present invention.

As used herein, the term “complex” refers to antibody-antigen or receptor-
Refers to the product of a specific binding reaction, such as a ligand reaction. A typical complex is an immune reaction product.

As used herein, “label” and “labeling means” (including grammatically various forms) refer to a single signal atom and molecule and are detectable signals that indicate the presence of a complex. Is directly or indirectly involved in the generation of. Any label or labeling means may be attached to or part of the expressed protein, polypeptide, antibody molecule of the invention, which is part of the antibody or monoclonal antibody composition of the invention, or separately. , Such atoms or molecules can be used alone or with additional reagents. Such labels themselves are well known in clinical diagnostic chemistry, but only so long as they are utilized in otherwise novel protein methods and / or systems form part of this invention. .

As the labeling means, a fluorescent labeling agent that chemically binds to an antibody or an antigen without denaturing the antibody or the antigen to give a fluorescent label can be used. This forms a fluorescent dye (dye) that is a useful immunofluorescence tracer. Suitable fluorescent labeling agents include fluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC), 5-dimethylamine-1-naphthalenesulfonyl chloride (DANSC), tetramethylrhodamine isothiocyanate (TRIT).
C), Lissamine, Rhodamine 8200 sulfonyl chloride (RB 200 SC
) Such as a fluorescent dye (dye). De for the description of immunofluorescence diagnostic technology
Luca, "Immunofluorescence Analysis," i
n Antibody As a Tool, Marchalonis et al., Ed
s. , John Wiley & Sons, Ltd. , Pp. 189-231
(1982). Its contents are incorporated herein by reference. Other suitable labeling agents are known to those skilled in the art.

In a preferred embodiment, the group of labels is horseradish peroxidase (H
RP), glucose oxidase and other enzymes. The main labeling group is HRP
Or an enzyme such as glucose oxidase, the receptor-ligand complex (
Additional reagents are required to visualize the fact that an immune reaction) has occurred. Such additional reagents for HRP include hydrogen peroxide, and oxidative dye precursors such as diaminobenzidine. An additional reagent useful in glucose oxidase is 2,2'-amino-di- (3-ethyl-benzthiazoliin-G-sulfonic acid) (ABTS).

Radioactive elements are also useful labeling agents and are used descriptively herein. Typical radioisotope labeling agents are radioactive elements that emit gamma rays. Elements that themselves emit gamma rays, such as ¹²⁴ I, ¹²⁵ I, ¹²⁸ I, ¹³² I, and ⁵¹ Cr, represent a gamma-emitting element production class. Particularly, ¹²⁵ I is preferable. Another group of useful labeling means are elements such as ¹¹ C, ¹⁸ F, ¹⁵ O and ¹³ N, which themselves emit positrons. The positrons thus emitted encounter the electrons present in the animal's body and produce gamma rays. Further useful are beta emitters such as Indium 111 or ³ H.

Labeling, ie, labeling polypeptides and proteins, is well known to those of skill in the art. For example, antibody molecules produced by hybridomas can be metabolically incorporated and labeled by providing the medium with amino acids containing a radioisotope (eg, Galfre et al., Meth. En.
zymol. , 73: 3-46 (1981)). Techniques for protein binding or coupling by activated functional groups are particularly applicable (eg, Aurameas et al., Scand. J. Immunol., Vol. 8 Su.
ppl. 7: 7-23 (1978); Rodwell et al., Biotech. Three
: 889-894 (1984); and U.S. Pat. No. 4,493,795).
.

The diagnostic system may also preferably include the specific binding agent as a separate package. A "specific binding agent" is a molecule that has the ability to selectively bind to the reagent species of the present invention or a complex comprising such a chemical species, but is not itself a molecular composition of a polypeptide or antibody of the present invention. A typical specific binding agent is complementary to a second antibody molecule, a protein or fragment thereof, S. aureus protein A and the like. Preferably, if the species is present as part of a complex,
The specific binding agent binds to the reagent species.

In a preferred embodiment, a label is attached to the specific binding agent. However,
If the diagnostic system contains an unlabeled specific binding agent, it is typically used as an amplification means, amplification reagent. In these embodiments, the labeled specific binding agent can specifically bind to the amplification means when the amplification means is attached to a complex containing the reagent species.

The diagnostic kit of the invention can be used in the "ELISA" format,
The amount of the polypeptide of the present invention in the sample is detected. "ELISA" refers to enzyme-linked immunosorbent assay using solid-bound antibody or antigen. Also, the enzyme antigen or enzyme antibody detects and quantifies the amount of the antigen in the sample. A description of the ELISA technology is provided by Sites et al., Basic and Clinical Immun.
topology, 4th Ed. , Lang Medical Publicat
ions, Los Altos, CA (1982) and US Pat. No. 3,654.
, 090; No. 3,850,752; and No. 4,016,043, the contents of which are incorporated herein by reference.

Thus, in some embodiments, a polypeptide, antibody of the invention or monoclonal antibody of the invention may be attached to a solid matrix of a solid support containing the package in a diagnostic system of interest.

The reagents are typically attached to a solid matrix by adsorption from an aqueous medium. However, other modes of conjugation addition applicable to proteins and polypeptides well known to those skilled in the art can be used. Typical adsorption methods are described here.

Useful solid matrices are well known to those skilled in the art. Such materials are insoluble in water, and are available from Pharma Fine Chemicals (Piscataway (NJ))
Cross-linked dextran available as EPHADEX ™; agarose; beads of about 1 micron to about 5 millimeters (mm) diameter polystyrene beads available from several suppliers (eg, Abbott Laboratories of North Chicago, IL). Nitrocellulose or nylon based webs (sheets, strings, spatulas) such as polyvinyl chloride, polystyrene, cross-linked polyacrylamide, sheets; tubes, plates or microtiter made from polystyrene or polyvinyl chloride Includes plates and wells.

The reagent species, labeled specific binding agent or amplification reagent of any diagnostic system described herein can be provided as a solution, a dispersion in a liquid, or as an essentially dry force. (For example, in lyophilized form). If the labeling means is an enzyme, the enzyme substrate can also be provided in a separate package of the system. As previously mentioned, solid supports such as microtiter plates and one or more buffers can also be included as separately packaged elements in the diagnostic assay system.

The packaging materials discussed herein with respect to diagnostic systems are those conventionally utilized in diagnostic systems. Having generally described the invention, it will be readily understood by reference to the following example. It should be noted that the following examples are provided for purposes of illustration and are not intended as limitations.

[0251]

【Example】

Example 1 Identification and Characterization of Polynucleotides Regulated by Neuroleptic Agents Male C57B1 / 6J mice (20-28 g) were divided into groups of 4 and treated with standard laboratory diet on a standard 12/12 h light / dark cycle. They were bred so that they could freely take tap water and tap water. In a typical experiment, mice were divided into 25 groups and treated as follows.

Control group: Mice were injected once with sterile saline (0.1 ml volume) or with no injection and sacrificed 45 minutes later. Acute neuroleptic treatment: In mice, the atypical neuroleptic clozapine (7
． 5 mg / kg) was injected once intraperitoneally. Mice were sacrificed 45 minutes later.

Chronic neuroleptic treatment: Mice were injected subcutaneously daily with clozapine (7.5 mg / kg) for 5 days to 2 weeks. All animals were sacrificed with CO ₂ in cages at designated times. The brain was quickly removed and placed on ice. The striatum including the nucleus accumbens was dissected and placed in ice-cold phosphate buffered saline.

The isolated RNA was prepared according to the method of Sutcliffe et al., P., incorporated herein by reference.
roc. Natl. Acad. Sci. USA, 97 (5): 1976-198.
1 (2000), International Patent Application WO 026406, US Patent No. 5,459,0.
37, US Pat. No. 5,807,680, US Pat. No. 6,030,784,
It was analyzed using the simultaneous sequence-specific identification method of mRNA known as TOGA (Total Gene Expression Analysis) described in US Pat. No. 6,096,503 and US Pat. No. 6,110,680. Preferably, RNA isolated by methods known in the art to form mRNA containing the starting poly A prior to applying TOGA technology.
Was concentrated. In a preferred embodiment, the TOGA method further included an additional PCR step performed using four 5'PCR primers in four different reactions and a cDNA template prepared from the antisense cRNA population. In the final PCR step using 256 5'PCR primers in different reactions, the first m
A PCR product was generated which was a cDNA fragment corresponding to the 3'region of the RNA population.
The resulting PCR product is then a) the remaining sequence of the recognition site of the restriction enzyme used to cleave and isolate the 3'region, and preferably four sequences immediately 3'to the rest of the recognition site. The first 5'sequence, including the base sequence syntax, preferably the complete fragment sequence,
And b) identified by the length of the fragment. Using these two parameters, sequence and fragment length, the resulting PCR products were compared to a database of known polynucleotide sequences. The length of the PCR product obtained was 5
Due to the inclusion of known vector sequences at the'and 3'ends, the sequences of the inserts listed in the sequence listing are shorter than the length of the fragments forming part of the digital address.

This method yields a digital sequence tag (DST), ie a polynucleotide representing the sequence tag at the 3'end of the mRNA. DSTs that show changes in relative concentration as a result of clozapine treatment were selected for further study. Labeled PCR
The intensity of laser-induced fluorescence of the products was compared for samples isolated from striatum / accumbens of mice treated with clozapine for 45 minutes, 7 hours, 5, 12, or 14 days.

In general, from poly (A) -enriched cytoplasmic RNA extracted from a tissue sample of interest, using an equimolar mixture or set of all 48 5'biotinylated anchor primers for initiating reverse transcription, Generate double-stranded cDNA. One such suitable set is G-A-A-T-T-T-C-A-A-C-T- to-G-G-A to A-G.
-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T
-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N (SEQ ID NO: 2
0) (V is A, C or G and N is A, C, G or T). One element of this 48 anchor primer mixture initiated the in situ synthesis of all copies of each mRNA species in the sample at the 3'end, thereby limiting the various 3'ends and biotinylated 2 A double-stranded cDNA is obtained.

Each of the biotinylated double-stranded cDNA samples was cut with the restriction enzyme MspI that recognizes the CCGG sequence. Then, the cDNA fragment obtained corresponding to the 3'end of the starting mRNA was biotinylated cD on a substrate coated with streptavidin.
It was isolated by capturing the NA fragment. Suitable streptavidin coated substrates include microtiter plates, PCR tubes, polystyrene beads, paramagnetic polymer beads and paramagnetic porous glass particles. A preferred streptavidin-coated substrate is a suspension of paramagnetic polymer beads (D
yinal, Inc. , Lake Success, NY).

After washing the streptavidin-coated substrate and the captured cDNA fragment,
C by digesting with NotI, which cuts an 8-nucleotide sequence that is present in the anchor primer but is rarely found in the mRNA-derived portion of cDNA
The DNA fragment product was released. 3'MspI of uniform length for each mRNA species
-NotI fragment so that the ClaI-NotI cut plasmid pBC SK ⁺ (Stratagen) is in the antisense orientation with respect to the T3 promoter of the vector.
e, La Jolla, CA) and used the product to Escheric
Hia coli SURE cells (Stratagene) were transformed. The ligation regenerates the NotI site and not the MspI site, releasing CGG, the first 3 bases at the 5'end of all resulting PCR products. Each library contained more than 5 × 10 ⁵ recombinants to ensure that the concentration of 0.001% or higher showed a high degree of variability in the 3 ′ end of total mRNA. A plasmid prep (Qiagen) was generated from the cDNA library of each sample studied.

An aliquot of each library was digested with MspI, which cuts at several sites in the parent vector but linearizes by releasing the flanking sequences, including the 3'cDNA insert and the T3 promoter. The product is T3 RNA
Incubation with a polymerase (MEGAscript kit, Ambion) generated an antisense cRNA transcript of the cloned insert containing the known vector sequences flanking the MspI and NotI sites of the original cDNA.

At this stage, each cRNA preparation was processed in a three-step procedure. In stage 1, c
250 ng of RNA was added to 5'RT primer (AGGGTTCGCGACG)
-G-T-A-T-C-G-G, (SEQ ID NO: 21) was used to convert to single-stranded cDNA. In step 2, 400 pg of the cDNA product was used as a PCR template for 4 different reactions, and the G-G-T-C-G-A-C-G-G-T-T-A-T-C-G-G-N (SEQ ID NO: 22), each of which is used with each of the four 5'PCR primers, and is a "generic"3'PCR primer G-A-G-C-T-C-C-A-C-C.
Paired with -G-C-G-G-T (SEQ ID NO: 23) to generate 4 sets of PCR reaction products ("N1 reaction products").

In step 3, the products of each subpopulation were further divided into 64 subsubpopulations (2 ng in 20 μl) for the second PCR reaction. For this PCR reaction, the "universal"3'PCR primer complexed with 6-FAM and fluoresceinated (SEQ ID NO: 23) and C-G-A-C-G-G-T-A-T-C-G was used. -Adding 100 ng of the appropriate 5'PCR primer in the form of GNNNNN (SEQ ID NO: 24) and minimizing artificial mispriming and promoting high fidelity replication It was included to use a program that included an annealing step at a temperature X just above the T _m of each 5'PCR primer. Each polymerase chain reaction step was performed in the presence of TaqStart antibody (Clonetech).

The products of the final polymerase chain reaction of each tissue sample (“N4 reaction product”).
) Was analyzed on a series of denaturing gels for DNA sequencing using an automated ABI Prizm 377 sequencer. GeneScan software package (
Data was collected using ABI) and amplitude and mobility were normalized. By completely carrying out this series of reactions, 64 subpopulations were produced for each of the 4 populations generated by the 5'PCR primers of the first PCR reaction, and the 5'PCR of the second PCR reaction was made. A total of 256 subpopulations were created with the entire primer.

The mRNA samples obtained at each time point were analyzed as described above. Table 1 is cD
It is the outline of the expression level of 495 mRNA determined from NA. These cds
NA molecules are identified by their digital address, the partial 5'terminal nucleotide sequence that binds the full length of the molecule, as well as the relative amounts of the molecule produced at different times after treatment. This partial 5'terminal nucleotide sequence is MspI (CC
GG) and the nucleotide sequence of the base syntax of the 5'PCR primer used in the final PCR step. The length of the fragment's digital address is determined by interpolating to a standard curve, which itself is determined by sequencing, so ± 1-2b. p. May fluctuate.

[0264] For example, the DNA identified by the digital address MspI AGTA.
The entries in Table 1 describing the molecules include the 5'terminal partial nucleotide sequence of CGGAGTA and 106b. p. It is further characterized by having a digital address of length. It is further described that the DNA molecule identified as MspI AGTA appears in increasing concentrations both after acute and chronic treatment with clozapine (see Figure 1). In addition, MspI AGTA 106
The DNA molecule identified as is represented by the nucleotide sequence corresponding to SEQ ID NO: 1.

Similarly, other DNs identified by MspI digital address in Table 1.
A molecule further includes 1) gene expression level in striatum / accumbens nucleus of mice not treated with clozapine (control), 2) gene expression in striatum / accumbens nucleus of mice treated with clozapine for 45 minutes. Concentration, 3) gene expression level in striatum / accumbens nucleus of mice treated with clozapine for 7 hours, 4) gene expression concentration in striatum / accumbens nucleus of mice treated with clozapine for 5 days, 5) with clozapine It is further characterized by striatum / accumbens gene expression levels in mice treated for 12 days, 6) striatum / accumbens nucleus gene expression levels in mice treated with clozapine for 14 days.

Several products shown differentially appeared to move to a position suggesting that this product was novel compared to the data excerpted from GeneBank. The sequences of such products were identified by one of two methods, cloning or direct sequencing of PCR products.

In addition, some of the isolated clones were further characterized as shown in Table 2 and their nucleotide sequences are shown in SEQ ID NOs: 1-19, 4 of the sequence listing below.
9-52, 57-72 and 107.

SEQ ID NOs: 1-19, 49-52, 57-72 and 107 are "5" of the sequence.
The addition of "C" had the MspI site found in the native state of the corresponding RNA shown. As described above, by linking the sequence to the vector M
spI is not regenerated. The experimentally determined sequence reported herein has CG as the first base at the 5'end.

The data presented in FIG. 1 shows that the 5'end is 6-carboxyfluorescein (6FA
M, ABI) paired with "universal"3'primer (SEQ ID NO: 23) 5
'PCR primer (C-G-A-C-G-G-T-A-T-C-G-G-A-
G-T-A, SEQ ID NO: 94). PCR reaction products were analyzed by gel electrophoresis on a 4.5% acrylamide gel and fluorescence data obtained on an ABI 1377 automated sequencer. Data were analyzed using GeneScan software (Perkin-Elmer). The sequence of the PCR product was determined using standard methods.

5'PCR with base syntax AGTA (SEQ ID NO: 94) showing PCR products generated from mRNA isolated from striatum / accumbens nucleus of mice treated with clozapine for various times as described above. The results of TOGA analysis using the primers are shown in FIG. The vertical axis index is about 160 b. p. PCR product of which the expression is increased as the striatum / accumbens of mice are treated with clozapine for 45 minutes, 7 hours, 5 days, 12 days, and 14 days.

Cloning of DST Without Candidate Partners and Validation of Cloning DST Using the Extended TOGA Method When appropriate, PCR products were isolated to obtain TOPO vector (Invitrog
en) and sequenced on both strands. Cloned DS
The database matches for each of the T sequences are listed in Table 2. An extended TOGA assay was performed on each DST to demonstrate that the cloned product corresponded to the TOGA peak of interest. PCR primers were designed based on the determined sequence, and PCR was performed using N1 PCR reaction product as a substrate. The oligonucleotide is a PCR product or DST cloned by extending the 3'end of the sequence G-A-T-C-G-A-A-T-C with a part of the MspI site (C-G-G). Was synthesized by adding 18 nucleotides adjacent to the determined sequence of For example, the digital address MspI AGTA 106 (SEQ ID NO: 1
5) PCR product is G-A-T-C-G-A-A.
-T-C-C-G-G-A-G-T-A-C-A-G-T-G-A-C-T-T
It was -TGAGT (SEQ ID NO: 28). This 5'PCR primer paired with a fluorescently labeled universal 3'PCR primer (SEQ ID NO: 23) in a PCR reaction using the PCR N1 reaction product as a substrate.

The length of the PCR product generated with the clone-specific primer (SEQ ID NO: 28) was compared to the length of the original PCR product generated by the TOGA reaction as shown in FIG. CLZ-3 (SEQ ID NO: 1) shows the PCR product generated with the clone specific primer (SEQ ID NO: 28) and the fluorescent labeled universal 3'PCR primer (SEQ ID NO: 23) in the upper panel (Figure 2A). There is. FIG. 2B shows the 5 ′ PCR primers C-G-A-C-G-G-T-A-T-C-G-G in the original TOGA reaction.
-A-G-T-A (SEQ ID NO: 94) and PCR products generated using fluorescently labeled 3'PCR primers. In the lower panel (Fig. 2C), the PCR products generated using the extended primers based on the cloned sequences are identical to the length of the original PCR products, with the top and middle panel graphics superimposed. Is shown. Other DST clones verified using this method include numerous cases (CLZ-5, SEQ ID NO: 2; CLZ-8, SEQ ID NO: 3; CLZ-.
10, SEQ ID NO: 4; CLZ-12, SEQ ID NO: 5; CLZ-15, SEQ ID NO: 6; C
LZ-24, SEQ ID NO: 7; CLZ-33, SEQ ID NO: 8; CLZ-34, SEQ ID NO: 9; CLZ-37, SEQ ID NO: 10; CLZ-38, SEQ ID NO: 11; CLZ-40.
, SEQ ID NO: 12; CLZ-6, SEQ ID NO: 14; CLZ-16, SEQ ID NO: 15; C
LZ-22, SEQ ID NO: 16; CLZ-32, SEQ ID NO: 17; CLZ-36, SEQ ID NO: 18; CLZ-42, SEQ ID NO: 19; CLZ-18, SEQ ID NO: 57; CLZ
-43, SEQ ID NO: 58; CLZ-44, SEQ ID NO: 59; CLZ-47, SEQ ID NO: 60; CLZ-48, SEQ ID NO: 61; CLZ-49, SEQ ID NO: 62; CLZ-5.
0, SEQ ID NO: 63; CLZ-51, SEQ ID NO: 64; CLZ-52, SEQ ID NO: 65
CLZ-56, SEQ ID NO: 67; CLZ-57, SEQ ID NO: 68; CLZ-60,
SEQ ID NO: 69; and CLZ-64, SEQ ID NO: 70). Table 3 contains the primers obtained from each of the cloned DSTs used in such studies. Direct Sequencing of PCR Products Obtained by TOGA and Validation by Extended TOGA Method In other cases, TOGA PCR products were sequenced using a modified direct sequencing method (Innis et al., Proc. Nat'l. Acad.Sci., 85
: 9436-9440 (1988)).

The PCR product corresponding to DST was gel purified and reamplified by PCR again to incorporate sequenced primers at the 5'and 3'ends. Addition of sequences was achieved by 5'and 3'ds-primers containing M13 sequencing primer sequences (M13 forward and M13 reverse, respectively) at the 5'end, followed by a linker sequence and a sequence complementary to the DST end. . Clontech Taq
Sterile H ₂ O, 10X PCR II buffer, dNT using the Start antibody system
P10 mM, MgCl ₂ 25 mM, AmpliTaq / antibody mixture (Taq antibody 1.1 μg / μl, AmpliTaq 5 U / μl), 5′ds-primer 10
0 ng / μl (5'TCC CAG TCA CGA CGT TGT AAA
ACG ACG GCT CAT ATG AAT TAG GTG ACC
GAC GGT ATC GG 3'SEQ ID NO: 89) and 3'ds-primer 100 ng / μl (5 'CAG CGG ATA ACA ATT TCA).
CAC AGG GAG CTC CAC CGC GGT GGC GGC
An original mixture was prepared containing all components except the gel-purified PCR product template containing C3 ', SEQ ID NO: 90). After adding the PCR template, PCR was performed using the following program. 94 ° C, 4 minutes and 94 ° C, 20 seconds; 65 ° C, 20
Sec; 72 ° C, 20 seconds 25 times, and 72 ° C, 4 minutes. The compatible PCR product obtained from the amplification was gel-purified as described above.

The purified ds-extended PCR product was sequenced using the standard protocol for ABI 3700 sequencing. Briefly, three pairs of forward and backward reactions were prepared and each reaction was gel purified ds-expanded N5 as template.
The PCR product was included. In addition, 2 μl of 2.5X sequencing buffer, Big D
ye Terminator mix 2 μl, 5 ′ sequencing primer (5 ′
CCC AGT CAC GAC GTT GTA AAA CG 3 ', SEQ ID NO: 91) or 3'sequencing primer (5'TTT TTT TTT TT
TTTT TTT V3 '(wherein V = A, C, or G, SEQ ID NO: 92) was included in a total amount of 10 μl.

[0275] In another embodiment, the 3'sequencing primer is the sequence 5'GGT GGC GG.
C CGC AGG AAT TTT TTT TTT TTT TTT TT
It was 3 '(SEQ ID NO: 93). PCR was performed using the following thermocycling program. 96 ° C., 2 minutes and 96 ° C., 16 seconds; 50 ° C., 15 seconds; 60 ° C. 29 times, 4 minutes.

The sequence of (CLZ-62, SEQ ID NO: 71 and CLZ-65, SEQ ID NO: 72) was determined by this method. Table 2 contains database data that matched the sequences determined by this method.

To demonstrate that the sequence was determined from the PCR product of interest by direct sequencing, the PCR primers were sequenced by direct sequencing as described above for sequences cloned into the TOPO vector. The PCR reaction was performed using N1 TOGA PCR reaction product as a substrate. Briefly, the oligonucleotide is G-A-T-C-G-A-A-.
The 3'end of the T-C sequence was extended with a partial MspI site (C-G-G) and synthesized by the addition of 18 nucleotides flanking the partial MspI site of the sequence determined by direct sequencing. This 5'PCR primer was used as a template for N1 TOGA.
In a PCR reaction with the PCR reaction product, it was paired with a fluorescently labeled universal 3'PCR primer (SEQ ID NO: 23). The length of these PCR products was compared to the length of the PCR product of interest. Table 3 contains the sequences of the primers used in these studies.

Demonstration of Candidate Candidates Using the Extended TOGA Method In four cases, the sequences listed in the TOGA PCR product, CLZ-17 (SEQ ID NO: 49), CLZ-26 (SEQ ID NO: 50), CLZ-28 (sequence No. 51) and CLZ-58 (SEQ ID NO: 52) were obtained from available Genbank, EST or candidate partners for sequences present in the appropriate databases. Table 4 lists candidates that were matched by accession number in the Genbank entry or by accession number for the EST assembled by the set of computers used to generate the consensus sequence.

To determine whether the TOGA PCR product of interest was obtained from the sequence of the candidate partner, the 3'end of the G-A-T-C-G-A-A-T-C sequence was segmented. M
PCR primers were designed by extending the spI site (C-G-G) and adding 18 nucleotides flanking the terminal MspI site of the candidate partner sequence. In the PCR reaction using the product of the first TOGA PCR reaction (N1 reaction product) as a template, each extended primer is combined with a fluorescently labeled universal 3'PCR primer (SEQ ID NO: 23). The PCR product obtained using the extended primer and the universal 3'primer was compared to the product obtained using the original TOGA PCR primer. The primers designed for such studies are shown in Table 4 along with the accession numbers of the sequences used to obtain the primer sequences.

Example 2 Characterization of CLZ5 (apoD) Another example of TOGA analysis is shown in FIG. In FIG. 3, the peak at about 201 is shown and when the 5'primer (SEQ ID NO: 25) was paired with SEQ ID NO: 23 to generate a list of PCR products, the digital address MspI CAC was obtained.
Identified as C 201. The PCR product was cloned and sequenced as described in Example 1. In order to prove the identity of the isolated clone (SEQ ID NO: 2), each of the candidates that extended the 3'end of the cloned sequence and added 12 to 15 nucleotides corresponds to the 5'PCR primer in the second PCR step. Was synthesized. In this case, the 5'PCR primer is G-A-T-C-G-A-A-T-C-C-G-G-C-A-C-C-T-.
It was A-C-T-G-G-A-T-C-C-T-TG-G (sequence number 29).
This 5'PCR primer paired with the fluorescently labeled 3'PCR primer (SEQ ID NO: 23) in PCR using the cDNA generated in the first PCR reaction as a substrate.

As shown in Tables 2 and 3, the CLZ-5 clone (CACC201) described above.
Is GenBank, which has been identified as mouse apolipoprotein D (apoD)
It corresponds to the k array X82648. Other corresponding apoD GenBan
The k sequences include L39123 (mouse), X55572 (rat), NM-001.
647 (human) are included. Northern blot analysis was performed to determine the effect of clozapine and haloperidol on apoD expression in the mouse striatum / accumbens nucleus. In situ hybridization analysis was also performed to determine the pattern of apoD expression in the striatum / accumbens of control and clozapine treated mice.

Male C57B1 / 6J mice (20-28 g) were divided into groups of 4 and housed on a standard 12/12 hour light / dark cycle with standard laboratory chow and tap water ad libitum. A typical experiment similar to that used in Example 1
Used for blot analysis. Briefly, sterile saline (0.1 ml volume)
Was injected once or the control group with no injection was sacrificed 45 minutes later. Haloperidol, a general neuroleptic drug for mice treated with acute neuroleptic drugs (4 mg / kg
) Or an atypical neuroleptic drug, clozapine (7.5 mg / kg), was injected once intraperitoneally and sacrificed after 45 minutes or 7 hours as described in Example 1. Haloperidol (4 mg / kg) injected subcutaneously daily for 10 or 14 days, or clozapine (7.5 mg / kg) injected daily for 5, 12, or 14 days,
Mice were chronically treated with neuroleptics. All animals were sacrificed with CO ₂ in cages at the indicated times. The brain was quickly removed and placed on ice. The striatum including the nucleus accumbens was dissected and placed in ice-cold phosphate buffered saline. Schibler et al., J
． Mol. Bio. , 142, 93-116 (1980), and homogenized tissues were extracted with phenol: chloroform to extract cytoplasmic RNA.
Was isolated. Poly A-rich mRNA was prepared from cytosolic RNA using a known method, oligo dT chromatography.

Northern blot analysis was performed using 2 μg of poly A-rich mRNA extracted from the striatum / accumbens nucleus of control and clozapine-treated mice, as shown in FIG. The mRNA transcript was electrophoresed on a 1.5% agarose gel containing formaldehyde and transferred to a biotrans membrane by the method of Thomas (Thomas).
mas, P.M. S. , Proc. Natl. Acad. Sci. , 77,5201
~ 5215 (1980)), 3 in Expresshyb (Clonetech)
Prehybridized for 0 minutes. CLZ-5 160 bp insert (25-100
ng) by [α- ³² P] -d CTP by the oligonucleotide labeling method to about 5 ×.
Labeling to 10 ⁸ cpm / μg was added to the prehybridization solution and incubated for 1 hour. Filter is high stringent (0.2 x SS
C) (1 X SSC: NaCl 0.015M, Na citrate 0.0015)
M) at 68 ° C., then Kodak X-AR film (Estman
Exposure to Kodak, Rochester, NY) for up to 1 week. Densitometry analysis of Northern blots was performed by ImageQuant software.

As shown in FIG. 4, a 900 bp mRNA consistent with the apoD gene was detected in control and treated mice. Expression of apoD mRNA is upregulated by clozapine treatment for 2 weeks. Clozapine may show antipsychotic effects by regulating apoD. Alternatively, in parallel with the mechanism of clozapine therapeutic effect, apoD may be co-regulated by clozapine and may be an indicator of clozapine bioactive concentration.

As shown in FIG. 5, Northern blot analysis was performed using mRNA extracted by the above method from striatum / nucleus accumbens of control mice and haloperidol-treated mice and the same ³² P-radiolabeled probe. A 900 bp mRNA corresponding to the apoD gene was detected in control and haloperidol treated mice. Interestingly, apoD mRNA expression is slightly downregulated by short-term and long-term haloperidol treatment. These results show that clozapine and haloperidol have different effects on apoD expression.

FIG. 6 is a graph comparing the results of clozapine-treated TOGA analysis of clone CLZ-5 (CACC201) shown in FIG. 4 and clozapine-treated Northern blot analysis of clone CLZ-5 shown in FIG. Northern blots were imaged with a phosphoimager to determine the amount of apoD mRNA in each clozapine-treated sample relative to the amount of control sample mRNA. As can be seen, the clozapine-treated TOGA assay shows a correlation with the clozapine-treated Northern blot assay.

Figures 7A-C demonstrate in situ the expression of apoD in mouse brain.
Hybridization analysis is shown. In situ hybridization is described in the literature (Thomas et al., J. Neurosci. Res.,
52, 118-124 (1998)) on suspension sections (25 μM thickness). Crown sections were hybridized for 16 hours at 55 ° C. with a ³⁵ S-labeled single-stranded 160 bp antisense cRNA probe of CLZ-5 (10 ⁷ cpm / ml). This probe is a 3'-terminal cDNA TOGA clone CLZ-
5 was synthesized using the Maxscript transcription kit (Ambion, Austin, Tex.). 2 × containing 14 mM β-mercaptoethanol
Excess probe was removed by washing with SSC (1 × SSC = 0.15M NaCl / 0.0015M sodium citrate) (30 min), then 0.5M NaCl / 0.05M EDTA / with 4 μg / ml ribonuclease. 0
． Incubated in 05M Tris-HCl (pH 7.5) at 37 ° C for 1 hour. In 0.5 × SSC / 50% formamide / 0.01M β-mercaptoethanol for 2 hours at 55 ° C., then in 0.1 × SSC / 0.01M β-mercaptoethanol / 0.5% sarkosyl, High stringency washing was performed at 68 ° C for 1 hour. The sections were fixed on gelatin-coated slides, dehydrated with ethanol and chloroform, and then autoradiographed. Slide the Ko
The dak X-AR film was exposed for 1-4 days and then dipped in Ilford K-5 emulsion. After 4 weeks, slides were developed with Kodak D19 developer, fixed and reverse stained with Richardson blue dye.

[0288] Figure 7A shows CLZ-5 (apoD) mRNA expression in the forebrain of mice, 7B shows apoD mRNA expression in the midbrain, and 7C shows expression in the hindbrain. In all brain sections, apoD is expressed by macroglia, buffy coat cells, perivascular fibroblasts and dispersed neurons. This is in agreement with previous studies investigating the expression of apoD in mice, rabbits and humans (Yoshida et al., DNA.
and Cell Biology, 15, 873-882 (1996); P.
rovost et al. J. Lipid Res. , 32, 1959-1
970 (1991); Navarro et al. , Neurosci. Le
tt. , 245, 17-20 (1998)).

The northern blot results (FIGS. 4 and 6) showed that apoD was induced by clozapine in the striatum of mouse brain. To investigate other sites of apoD induction, in situ hybridization analysis was performed on brains from saline-treated and clozapine-treated mice. 8A-I are in s
5 represents the in situ hybridization analysis, carried out by the method described above, in saline treatment (top row) or clozapine (7.5 mg / kg) for 5 days (middle row) and 1
3 shows clone CLZ-5 (apoD) mRNA expression in mouse forebrain (8A-C), midbrain (8D-F) and hindbrain (8G-I) after 4 days treatment (bottom row). The animals were killed by intracardiac perfusion of 4% paraformaldehyde, the brains were removed, post-fixed for 12 hours, cryoprotected with 30% sucrose and snap frozen at -70 ° C. At low magnification, striatum, cortex, pallidum (GP) both on day 5 and on week 2 of clozapine treatment.
) And an increase in apoD mRNA in the thalamus. Increased apoD expression was also detected in the white matter tract, predominantly in the corpus callosum (cc), anterior commissure, internal capsule (ic) and optic tract (opt). Although cells with increased apoD expression were also seen at high magnification, enhancement of the apoD hybridization signal in the striatum, pallidus, and thalamus mainly increased the number of cells expressing detectable apoD. It was clear that this was because of the

Immunohistochemical analysis was performed to assess changes in apoD protein expression in response to clozapine using monoclonal antibodies raised against full length apoD. Increased protein expression correlated well with increased mRNA expression (data not shown). Combined in situ hybridization and immunohistochemical studies, elevated levels of apoD are mainly localized to various white matter tract neurons and striatal astrocytes and oligodendrocytes throughout the brain. Proved to do.

9A-H show corpus callosum (cc, FIG. 9A, E); putamen caudate (CPu, FIG. 9B, 7).
F); anterior commissure (aca, FIG. 9C, 9G); and pallidum (GP, FIG. 9D, 9H).
3 shows dark field photographs demonstrating that apoD mRNA expression was upregulated in various brain regions including. In situ hybridization was performed with ³⁵ S-labeled antisense apo D riboprobes as described above on brains from control animals (FIGS. 9A-D) and clozapine-treated animals (FIGS. 9E-H). The observed up-regulation of apoD was due to increased apo expression per cell.

10A, B show dark field photographs demonstrating that apoD mRNA expression was upregulated in the encapsulation (ic). 10C, D show brightfield photographs of the optic optic optics demonstrating that apoD expression was upregulated in oligodendrocytes. In situ hybridization was performed with ³⁵ S-labeled antisense apo D riboprobes as described above on brains from control animals (FIGS. 10A, C) and clozapine treated animals (FIGS. 10B, D). As shown in FIG. 10D, cells that express apoD predominantly in the optic tract have a box-like morphology, and are arranged in a continuous array, probably along the axon. Such a feature is characteristic of oligodendrocytes that synthesize the insulating myelin coat of nerve fibers. In brain performed from haloperidol-treated mice.
In situ hybridization experiments did not show a substantial increase in apoD expression in gray matter or white matter areas (data not shown).

White matter tracts contain nerve fiber bundles that connect different regions of the brain. The major cells in these regions are astrocytes and oligodendrocytes, both of which have been shown to express apoD (Boyles et al., J. Lipid Res.
． , 31, 2423-2256 (1990); Navarro et al. ，
Neurosci. Lett. , 245, 17-20 (1995); Provo.
st et al. J. Lipid Res. 32, (1991)). Antibodies specific for the astrocyte marker glial fibrillary acidic protein (GFAP) or oligodendrocyte marker to determine which cell type is involved in increased apoD signaling 2 Antibody specific for ', 3'-cyclic nucleotide 3'-phosphodiesterase (CNP) (Boehringer Mannhei)
m, Germany) in combination with a ³⁵ S-labeled apoD riboprobe. Vectastain ABC ™ Kit (Vect
or Laboratory Inc., Burlingame, Calif.) was used according to the manufacturer's instructions to detect immune responses.

Floating sections of brain were treated with blocking solution (0.1% Triton X-100 / PB).
4% bovine serum albumin in S) for 2 hours at room temperature and then with anti-GFAP or anti-CNP antiserum (1: 500 dilution) in blocking solution for 16-20 hours at 4 ° C. The section is then 0.1% Trit
Washed with on X-100 / PBS and incubated with secondary biotinylated antibody (1: 200 dilution in blocking solution) for 2 hours at room temperature. The section is then cut to 0.
Wash with 1% Triton X-100 / PBS, incubate with ABC reagent (1: 1 dilution in blocking solution) for 1 hour and finally 0.1% Triton.
It was washed with X-100 / PBS. Enzyme expression was performed in 0.05% diaminobenzene in PBS containing 0.003% hydrogen peroxide for 3-5 minutes.

FIG. 11 shows striatum and optic cord sections of control and clozapine-treated animals. The thick arrow indicates co-localization of GFAP and apoD, and the thin arrow indicates expression of apoD only. 11A, B show that in the untreated striatum, a large number of GFAP-positive cells are apoD-positive in both gray matter and white matter areas. 11D and 11E,
It is shown that in the brain obtained from clozapine-treated animals, an increase in the amount of apoD was observed in some GFAP-positive cells in the striatum. Furthermore, the number of non-GFAP-positive cells expressing apoD was increased in the striatum and in the pallidum and thalamus. These appear to be neurons based on size and morphology.

FIGS. 11C, F show co-localization of GFAP and apoD in the optic tract of control animals (11C) and clozapine treated animals (11F). Although some astrocytes express apoD mRNA, the cells involved in the major upregulation of apoD transcripts are not labeled with GFAP and therefore appear to be oligodendrocytes. Non-GFAP-expressing cells expressing apoD in other areas of the white matter, such as the corpus callosum, anterior commissure and encapsulation, appear to be oligodendrocytes. However, expression in microglial cells cannot be ruled out. 11G, H are apoD immunizations with anti-human-apoD primary antibody (Novocastra, Newcastle, UK) in the visual tracts of saline-treated control animals (11G) and clozapine-treated animals (11H). Shows histochemical reaction.

Co-localization studies performed with anti-CNP antibodies showed CNP immunoreactivity in the white matter tracts throughout the central nervous system that correlates with regions of the apoD mRNA hybridization signal. This indicates that apoD was expressed in oligodendrocytes. However, within the striatum gray matter region, there was no co-localization consistent with neuronal apoD accumulation (data not shown).

FIG. 12 shows Northern blot analysis of clone CLZ-5 expression in cultured glial cells treated with clozapine (100 nM and 1 μM) for 1 or 7 days. Glial cell cultures were prepared from postnatal day 2 rats. After treating cells with different concentrations of clozapine for different lengths of time, mRNA was extracted according to the following: A = control (no clozapine); B = 100 nM clozapine, 1 day; C = 1 μ.
M clozapine, 1 day; D = 100 nM clozapine, 1 week; E = 1 μM clozapine, 1 week. 20 μg of total cytoplasmic RNA from glial cultures was electrophoresed on a 1.5% agarose gel containing formaldehyde, blotted and tested as described above. Interestingly, 1 for clozapine (both 100 nM and 1 μM)
ApoD mRNA levels were down-regulated in weekly treated mixed glial cultures. This would suggest that neurons and glial cells have different mechanisms for apoD regulation.

[0299] TOGA, Northern blot analysis and in situ hybridization studies showed increased apoD mRNA expression in response to long-term clozapine administration in both white matter and gray matter regions of the mouse brain. Colocalization studies combining in situ hybridization and immunohistochemistry showed that administration of clozapine resulted in apoD mR in both mouse neurons and glial cells in mice.
It becomes clear that the NA level has increased. This evidence indicates that the glial cells responsible for the most significant increase in apoD expression are predominantly oligodendrocytes, but some astrocytes also had increased apoD expression after clozapine treatment. . In contrast, TOGA, Northern blot and in situ hybridization analysis showed that apoD expression was not affected by haloperidol treatment.

In addition to the above mouse studies showing that apo D is regulated by long-term administration of antipsychotics, in human subjects with schizophrenia and bipolar disorder, in the prefrontal cortex of these patients. It was shown that apoD expression is increased. Taken together, these results suggest that apoD is a neuropathological marker associated with mental disorders and can be used to target abnormalities in specific brain anatomical regions.

Apo D was initially identified as a component of plasma high density lipoprotein (HDL) which also contains phospholipids, cholesterol and fatty acids (McConathy).
et al. , Fed. Eur. Biochem. Soc. Lett. , 37:
178 (1973)). Apo D is thought to play a role in reverse cholesterol transport in blood, the removal of excess cholesterol from tissues to the liver for catabolism (Oram et al., J. Lipid Res., 37 :( 1
996)). Apo D is abundantly expressed in human serum and is the major protein component of cystic fluid obtained from women with human breast cystic disease (Balbin et al.
al. , Biochem. J. , 271: 803 (1990)), liver, kidney,
Widely expressed in many tissues including intestine, spleen and brain (Drayna et.
al. J. Biol. Chem. , 261: (1986)). In the human central nervous system, as in other species (Provost et al., J. Lipid R.
es. 32: ((1991); Seguin et al., Mol. Bra.
in Res. , 30: 242 (1995); Smith et al. J.
Lipid Res. , 31: 995 (1990)) apoD is mainly glial cells,
Expressed in buffy coat cells, perivascular cells, and some neuronal populations (Na
varro et al. , Neurosci. Lett. , 254, 17 (1
995); Kalman et al. , Neurol. Res. , 22:33
0 (2000)). The physiological role of apoD in the central nervous system is unknown, but it has been shown to bind several hydrophobic ligands including sterols and steroid hormones (Dilley et al., Breast Canc. R.
es. Treat. 16: 253 (1990); Lea, O .; A. , Ster
Oids, 52: 337 (1988)). This suggests a role in extracellular lipid transport in the brain. Apo D has also been shown to bind arachidonic acid (M
orais-Cabral et al. , FEBS Lett. , 366: 5
3 (1995)), which is implicated in functions associated with cell membrane remodeling and prostaglandin synthesis. In the process of sciatic nerve regeneration, a process involving significant membrane degradation and lipid release, apoD levels are increased by 500-fold (Boyle
s et al. J. Biol. Chem. , 265: 17805 (1990)
)). In a recent report, the rat olfactory cortex (entorhi)
nal cortex) and increased apoD expression after experimental and chemical lesions of the hippocampus (Ong et al., Neurosci., 79).
: 359 (1997); Terisse et al. , Mol. Brain
Res. , 70:26 (1999)). Furthermore, in humans, apoD accumulates in the cerebrospinal fluid and hippocampus of patients with Alzheimer's disease and other neurological disorders (Te
risse et al. J. Neurochem. , 71: 1643 (19
98)). Thus ApoD may be functioning in a pathological condition,
Alternatively, expression of apoD may be an indication of efforts to compensate for the neuropathological conditions associated with such injury.

The apoD expression pattern in the brain suggests that apoD has an important role in psychotic disorders. It is generally believed that imbalances in the basal ganglion circuit are associated with psychotic behaviour, and blockade of specific receptors in these areas is involved in neuroleptic action. ApoD in neurons of striatum and pallidum
Increased mRNA expression is consistent with this hypothesis.

Furthermore, the apoD induction seen in the encapsulation is of particular interest. The inner capsule consists of numerous nerve fibers that connect the thalamus to the cortex, and is the region where the fiber tracts that cross the striatum converge. The thalamus is the place of transfer of virtually all information transmitted to the cortex, and coordinated cortico-thalamic activity is essential for normal consciousness. Recent theories link psychotic behavior with perturbation of cortical-thalamic oscillations. Upregulation of apoD expression in the encapsulation appears to play a role in restoring the proper balance of neural communication.

Furthermore, aberrant lipid neurochemical states caused by aberrant lipid transport and metabolism have been associated with psychotic disorders such as schizophrenia (Walker et al., B.
r. J. Psych. , 174, 101-104 (1999)). Numerous reports link psychotic disorders with disorders of cholesterol metabolism and indicate psychosis as C-type Niemann-
It is described as an early symptom of Pick's disease (Campo et al., Dev
elop. Med. and Child Neurol. , 40: 126-12
9 (1998); Shulman et al. , Neurology, 45,
1739-1743 (1995); Turpin et al. , Dev. Ne
urosci. , 13, 304-306 (1991)). Niemann-Pick disease is an autosomal recessive disorder associated with abnormal cholesterol metabolism (Yoshid
a et al. , DNA and Cell Biology, 15,873
-882 (1996)). Other reports suggest that myelin dysfunction can cause mental illness. If most of the brain's cholesterol is taken up by myelin, abnormal cholesterol metabolism can lead to myelin dysfunction. Since myelin acts as an insulator along nerve axons, action potentials are rapidly propagated along nerve fibers. It has been hypothesized that molecular abnormalities in myelin cause dysregulation of neuronal connectivity, which is responsible for mental illness (Weikker).
t et al. , Schizophrenia Bull. , 24,303-
316 (1998)).

Although the physiological function of apoD in the central nervous system is not clear, several lines of evidence suggest a role for apoD as a vehicle for extracellular lipid transport and lipid transfer (particularly for cholesterol) in the nervous system. Is suggested. Apo D is a component of plasma high density lipoprotein (HDL) that also contains phospholipids, cholesterol and fatty acids. Although less is known about HDL compared to other plasma lipoproteins LDL and VLDL, it is generally believed that HDL protects against cardiovascular disease by removing excess cholesterol from cells of the arterial wall. .
This elimination involves a direct interaction of HDL lipoproteins with the plasma membrane domain and subsequent transport to the liver for catabolism (Oram et al., J.
． Lipid Res. , 37: 2473-2491 (1996)). further,
Apo D is synthesized and secreted by cultured astrocytes and this secretion has been shown to be increased in the presence of cholesterol derivatives (Patel et al.
, Neuroreport, 6,653-657 (1995)). Furthermore, it was also proved that the apoD level was elevated in Niemann-Pick type C disease accompanied by the elevation of cholesterol level. These studies provide evidence that apoD has a functionally important role in central nervous system cholesterol transport.

In addition to studies investigating the correlation between cholesterol levels and psychotic behavior, other studies have found a correlation between cholesterol levels and treatment with neuroleptic drugs. For example, as far back as the 1960s, there are reports demonstrating elevated serum cholesterol in patients treated with commonly used neuroleptic drugs such as chlorpromazine and haloperidol (Spivak et al., Clin.
Neuropharm. , 22, 98-101 (1999); Fleischh.
acker et al. , Pharmacopsy Chitry, 19, 1
11-114 (1986); Clark et al. , Clin. Pharm
． and Therapeutics, 11, 883-889 (1970)).
. However, newer atypical antipsychotics such as fluperlapin (flu
perlapine) and clozapine did not show a similar rise (
Spivak et al. , Clin. Neuropharm. , 22, 98
-101 (1999); Fleischhacker et al. , Phar
macopsychiatry, 19, 111-114 (1986); Bost
on et al. , Biol. Psych. , 40, 542-543 (199
6)). Interestingly, the results of the present invention revealed that clozapine and haloperidol have different effects on apoD expression. This may explain the differences seen in cholesterol regulation. Although the mechanism for these cholesterol changes is not known, the data of the present invention show that when the neuroleptic-induced changes in apoD expression and the cholesterol-binding ability of apoD are combined, the neuroleptic-induced changes in cholesterol levels are shown. Suggest that can be explained.

In addition to research on cholesterol trends, there is a paper focusing on the link between metabolic disturbances of phospholipids and fatty acids and mental disorders (for a review, see Horrobin e.
t al. , Prostaglandins, Leukotrienes an
d Essential Fatty acids, 60, 141-167 (1
999))). For example, numerous studies have reported differences in total membrane phospholipid content, fatty acid levels, cholesterol levels and cholesteryl ester levels in fibroblasts and / or frontal cortex of patients with schizophrenia (Kesha.
van et al. J. Psychiatry Res. , 49, 89-9
5 (1993); Mahadik et al. , Schizophrenia
Res. , 13, 239-247 (1994); Sengupta et a.
l. , Biochem. Med. 25, 267-275 (1981); Ste
vens et al. , Schizophr. Bull. , 6,60-61 (
1972)). Membrane phospholipids act as precursors to many signal transduction pathways (eg, inositol phosphates, arachidonic acid, platelet activating factors, and eicosanoids) and constitute the membrane environment for neurotransmitter-mediated signal transduction. . Therefore, altered membrane phospholipid metabolism can have serious consequences for nerve traffic and can lead to behavioral abnormalities.

Changes in the structure and function of cell membranes may be caused by changes in the content and distribution of lipids and fatty acids in the membrane, such as arachidonic acid. Arachidonic acid is released by the action of numerous phospholipase enzymes, primarily phospholipase A2, and is a substrate for the synthesis of prostaglandins and leukotrienes. Although the molecular mechanisms responsible for complex phospholipid biochemical abnormalities are unknown, several groups have demonstrated enhanced phospholipase A2 activity in the plasma and brain of schizophrenic patients (Gattaz).
et al. , Biol. Psychiatry, 22, 421-426 (1
987); Ross et al. Arch. Gen. Psychiatry
, 54, 487-494 (1997); Ross et al. , Brain
Research, 821, 407-413 (1999)). Furthermore, plasma phospholipase A2 levels have been shown to be reduced after neuroleptic therapy (Gatta).
z et al. , Biol. Psychiatry, 22, 421-426 (
1987)). Other molecular candidates suggested to be involved in mental illness include phospholipase C enzyme, diacylglycerol kinase, and inositol phosphate (Horrobin et al., Prostaglandins,
Leukotrienes and Essential Fatty aci
ds, 60, 141-167 (1999)).

Interestingly, apoD was shown to bind cholesterol as well as arachidonic acid specifically. ApoD is an atypical apolipoprotein in that it has no sequence homology with other apolipoproteins (Weech et al.
al. , Prog. Lipid Res. , 30, 259-266 (1991)
), Rather a protein of the lipocalin superfamily. The latter has the function of transporting small hydrophobic molecules including sterols, steroid hormones and arachidonic acid (Balbin et al., Biochem.
． J. , 271, 803-807 (1990); Diley et al. ，
Breast Cancer Res. Treat. , 16: 253-260 (
1990); Lea, Steroids, 52: 337-338 (1988);
Boyles et al. J. Lipid Res. , 31,2243-2
256 (1990)). As a lipid-binding protein, apo D affects fatty acid composition, cholesterol levels, and membrane phospholipids. All of these affect the composition and structure of cell membranes. Since apo D specifically binds cholesterol, arachidonic acid and other lipids, changes in apo D levels also affect lipid metabolism and signal transduction by affecting the substrate availability of these pathways.

Another finding suggesting that ApoD is involved in psychosis is the finding that ApoD may have a chromosomal association with schizophrenia. The position of apo D on the chromosome is 3q2
It is 6. Although its association is relatively indeterminate, genetic studies have suggested a potential association between schizophrenia and chromosome 3q (Maier et al., Curr. Opin.
． Psych. , 11, 19-25 (1998)).

Northern blot analysis of striae from haloperidol-treated mice did not show the same increased apoD expression as clozapine. Schizophrenia is a heterogeneous disorder that includes multiple subtypes. The differences in clinical effect between clozapine and haloperidol are thought to reflect different schizophrenic subtypes with different pathways and mechanisms. Therefore, regulation of apoD may be a unique mechanism of action of clozapine.

Subtypes of serotonin in this regard, such as 5HT _2a and 5HT _2c are
It appears that clozapine may provide the pharmacological mechanism of action on apoD expression. From the results obtained so far, ketanserin and mesulergine, which are selective antagonists of 5HT _{2a / 2c} and 5HT _2c , respectively.
Gine) is shown to clearly upregulate the expression of apoD mRNA in the mouse brain. Striatum is known to express multiple 5HT receptor subtypes, including 5HT _2c, which appears to be the subtype that mediates the effect of clozapine on apoD expression. In contrast,
Cultured glial cells or astrocytes do not appear to express the 5HT _2c receptor. Thus, the down-regulation seen in these cells may reflect actions at different subtypes, such as 5HT _2a , or at different receptors. Furthermore, in studies of hypertension, ketanserin has been associated with lower total cholesterol levels and upregulation of other apolipoprotein apoA1 (Loschiavo et al., Int. J. Cli.
n. Pharmacol. Ther. Toxicol. , 28,455-457
(1990)). The similar effects seen on both ketanserin and clozapine suggest that they operate via the same receptor subtype (one or more).

The finding that apoD mRNA levels are elevated by clozapine correlates the mechanism of action of apolipoprotein and neuroleptic drugs. Combined with the recent evidence that schizophrenia and other neuropsychiatric disorders are associated with abnormal lipid metabolism, apoD has an important role in the action of clozapine. It is suggested.

Example 3 CLZ-40 Elucidation Male C57BI / 6J mice (20-28 g) were housed as previously described in Example 1. The same experimental method used in Example 1 for clozapine treatment was used for the various analyzes below. That is, in the clozapine test, mice in the control group were given a single injection of sterile physiological saline (volume 0.1 ml) or not injected and killed 45 minutes later. Short-term clozapine-treated mice were given a single intraperitoneal injection of clozapine (7.5 mg / kg) as described in Example 1 and killed 45 minutes or 7 hours later. Mice treated with long-term clozapine were given an intraperitoneal injection of clozapine (7.5 mg / kg) once daily for 5 days, 12 days or 14 days as described in Example 1. CO ₂ in all of these animals at the specified time in the cage
Killed in The brain was immediately removed and placed on ice. Cut off the filament including the nucleus accumbens,
Place in ice-cold phosphate buffered saline. MR according to the method described in Example 2.
NA was prepared.

For the morphine test, male C57BI / 6J mice (20-28 g) were housed as described previously in Example 1 and divided into the following groups: 1) Control group: mice were anesthetized with halothane. Placebo pellet was subcutaneously implanted; 2) Short-term morphine treatment group: mice were intraperitoneally injected with morphine 10 mg / kg; 3) Long-term treatment or resistance group: 1 pellet containing morphine free base 75 mg subcutaneously The mice were tolerated and addicted to the drug by implanting in mice for 3 days; 4) Withdrawal group: naltrexone (naltrex) in morphine-resistant mice.
one) 1 mg / kg was intraperitoneally injected.

72 hours after placebo or morphine pellet implantation, or 4 hours after single morphine injection, or 4 naltrexone administration to morphine resistant mice.
After hours, the animals were killed by CO ₂ in cages. The brain was promptly removed. The striatum including the nucleus accumbens and the tissue mass including the amygdala were cut out under a microscope and ice-cooled R
Collected in NA extraction buffer.

The TOGA data shown in Figures 13 and 14 show that 5'-PCR paired with a "universal"3'-primer (SEQ ID NO: 23) 5'end labeled with 6-carboxyfluorescein (6FAM, ABI). Primer (C-G-A-C-G-G-T-A
-T-C-G-G-T-T-G-T; SEQ ID NO: 26). PC
R reaction products were separated by gel electrophoresis on a 4.5% acrylamide gel,
Fluorescence data was obtained on an ABI377 automated sequencer. Data were analyzed using GeneScan software (Perkin-Elmer).

Parsing nucleotide sequence C-G-A-C-G-G-T-A-T-C-G-G-T-
The results of TOGA analysis using the 5'PCR primer with TGT (SEQ ID NO: 26) are shown in Figures 13 and 14. These represent PCR products prepared from mRNA isolated from striatum / accumbens of mice treated with clozapine (Figure 13) or morphine (Figure 14). In FIG. 13, the vertical indicator line indicates the approximately 266 bp PCR product present in control cells. It was expressed by clozapine for 45 minutes, 7
There is a decrease in the striatum / accumbens of mice treated for hours, 5 days, 12 days and 14 days. CLZ-40 down-regulation already occurs 45 minutes after clozapine treatment and remains down-regulated for at least 14 days.

In FIG. 14, the vertical indicator line indicates the approximately 266 bp PCR product present in control cells. Its expression is control striatum (PS), short-term treatment striatum (AS), withdrawal striatum (WS), control amygdala (PA), short-term treatment amygdala (AA), long-term treatment amygdala (T).
A), and in the withdrawn amygdala (WA) under different regulation. CLZ-40 product expression is greater in the striatum than in the amygdala. Furthermore, CLZ-40 exhibits long-term treatment-specific or withdrawal-specific regulation in both of these brain regions. In the striatum, CLZ-40 is downregulated in the detachment striatum but not in the short-term treatment striatum. In the amygdala, CLZ-40 is slightly upregulated in short-term treated amygdala and is gradually upregulated in long-term treated and absent amygdala.

Northern blot analysis was performed using mRNA extracted from the striatum / accumbens nucleus of control and clozapine-treated mice, as shown in FIG. Thus, an agarose gel containing 2 μg of poly A-enriched mRNA and a size standard was electrophoresed on a 1.5% agarose gel containing formaldehyde, transferred to a biotransmembrane and transferred in Expreeshyb (Clonetech) for 30 minutes.
Pre-hybridized for a minute. 265 bp CLZ-40 insertion sequence (
25-100 mg) was labeled with [α- ³² P] -dCTP by the oligonucleotide labeling method.
Was labeled with a specific activity of about 5 × 10 ⁸ cpm / μg, added to the prehybridization solution and incubated for 1 hour. The filter was washed under high stringency at 68 ° C. (0.2 × SSC) (1 × SSC: 0.015M NaCl and 0.0015).
M sodium citrate) followed by Kodak X-AR film (Eastma)
n Kodak, Rochester, NY) for up to 1 week. Figure 15
As shown in, a 9-12 Kb transcript was detected in control and clozapine-treated mice. It drops sharply after 45 minutes for clozapine treatment and remains down-regulated for at least 14 days.

FIG. 16 is a graph comparing the results of clozapine-treated TOGA analysis of clone CLZ-40 shown in FIG. 13 and clozapine-treated Northern blot analysis of clone CLZ-40 shown in FIG. Northern blots were imaged by phosphoimager to show CLZ-4 of each clozapine-treated sample relative to the mRNA level of the control sample.
The amount of 0 mRNA was measured. As you can see, clozapine treated TOGA
The analysis shows correlation with clozapine-treated Northern blot analysis.

17A-B show in situ hybridization analysis demonstrating expression of CLZ-40 mRNA in mouse brain. In situ hybridization was performed on suspension sections (25 μM thickness). 5 crown sections
It was hybridized with a ³⁵ S-labeled single-stranded antisense cRNA probe of CLZ-40 (10 ⁷ cpm / ml) at 5 ° C. for 16 hours. This probe is 3 '
-Terminal cDNA From TOGA clone from Maxscript transcription kit (Am
bion, Austin, Texas). 2 × SSC containing 14 mM β-mercaptoethanol (1 × SSC = 0.15M NaCl
/0.0015M sodium citrate) to remove excess probe (30 minutes), then 0.5M Na with 4 μg / ml ribonuclease.
3 in Cl / 0.05M EDTA / 0.05M Tris-HCl (pH 7.5)
Incubated at 7 ° C for 1 hour. 0.5 × SSC / 50% formamide / 0.
In 01M β-mercaptoethanol for 2 hours at 55 ° C., then 0.1 × SSC
/0.01M β-mercaptoethanol / 0.5% sarcosyl at 68 ° C 1
High stringency washing was performed for a period of time. The sections were fixed on gelatin-coated slides, dehydrated with ethanol and chloroform, and then autoradiographed. The slides were exposed to Kodak X-AR film for 1-4 days and then Ilford
It was immersed in a K-5 emulsion. After 4 weeks, slides were developed with Kodak D19 developer, fixed and reverse stained with Richardson blue dye. Interestingly, CLZ-40 mRNA is specifically expressed in the nucleus accumbens and piriform cortex (FIG. 17A) and dentate gyrus (FIG. 17B), but not detected in other areas of the brain.

Currently, the identity of CLZ-40 (SEQ ID NO: 12) is unknown. But CLZ
When PCR amplification of -40 DST was performed, as shown in Table 4, the extended sequence clone of CLZ-40 (SEQ ID NO: 13) was identified as EST (AI5 of GenBank database).
09550). The finding that CLZ-40 is downregulated by clozapine treatment suggests an association with the therapeutic effect of clozapine. Moreover, its highly unique gene expression pattern is unlike any other gene identified to date, and its presence in the nucleus accumbens suggests that CLZ-40 has many functional roles associated with this structure. , That is involved in limbic / mental behavior and addiction.

Addiction to narcotics and other drugs of abuse is a chronic disease of the brain, most likely caused by the molecular and cellular adaptation of certain neurons to repeated exposure to narcotics (Leshner, A. , Science, 278, 45-47 (
1997)). An important neural substrate that is thought to be involved in the potentiating action and addiction of narcotics is the mesolimbic system, especially the nucleus accumbens (Everitt et al., Ann. N. et al.
Y. Acad. Sci. , 877, 412-438 (1999)). Highly addictive drugs such as opium, cocaine and amphetamine cause adaptation to the neural circuit of the nucleus accumbens, but the exact relationship is unknown. The molecular neuroadaptation that occurs in this structure will provide important insights into the mechanism of drug addiction. C
Since LZ-40 is specifically expressed in the nucleus accumbens, it is a candidate that may be involved in such a mechanism. The elucidation of the biological states responsible for psychosis and addiction is key to understanding the cause of such disorders and will lead to the development of more effective treatments, including anti-addiction drugs.

Moreover, behavioral mechanisms associated with addiction reflect learning and memory mechanisms (Whi
te, N.E. , Addition, 91, 921-949 (1996)). The hippocampal system has long been associated with learning and memory (Sziklas et
al. , Hippocampus, 8, 131-137 (1998)). This includes conditional associative learning (conditional associative learning).
A form of associative learning is included (Di Chi
ara et al. , Ann. N. Y. Acad. Sci. , 877,461
-85 (1999)). The expression of CLZ-40 in the hippocampus may be that this gene provides a link with such learning processes.

[0326]

[Table 1]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[Table 2]

[0339]

[0340]

[0341]

[0342]

[0343]

[Table 3]

[0344]

[0345]

[0346]

[0347]

[0348]

[Table 4]

[0349]

[0350]

[Table 5]

Example 4 Characterization of CLZ 34 Male C57B1 / 6J mice (20-28 g) were housed as described above in Example 1. The same experimental paradigm used in Example 1 for clozapine treatment was used for the TOGA analysis shown below.

The TOGA data shown in FIG. 18 shows that the 5'end is 6-carboxyfluorescein (6FAM,
5 paired with "universal"3'primer (SEQ ID NO: 23) labeled with ABI)
It was obtained using the'-PCR primer (CGACGGTATCGGTATT; SEQ ID NO: 27). PCR reaction products were electrophoretically separated on a 4.5% acrylamide gel, and fluorescence data were obtained on an ABI 377 automated sequencer. Data is GeneScan software (Pe
rkin-Elmer).

As described in Example 1, the analytic base CGACGGTATCGGTATT (representing the PCR product resulting from mRNA isolated from striatal / nuclear accumbens of mice treated with clozapine for various lengths of time. 5'PCR of SEQ ID NO: 27)
The results of TOGA analysis using the primers are shown in FIG. In FIG. 18, a vertical indicator line is present in control cells and its expression in striatum / nuclear accumbens of mice treated with clozapine is specific for acute versus chronic treatment. Shows a PCR product of about 89 b.p. CLZ-34 increased after 45 minutes and 7 hours of clozapine treatment but decreased to control levels by day 5 and remained at control levels by day 12 with some slight increase on day 14 There was an increase. In situ analysis performed using CLZ-34 as a probe revealed that CLZ-34 is expressed throughout the brain (data not shown).

Rat N-methyl-D-aspartate receptor / NMDAR1-2a subunit (NMDAR1)
CLZ-34 corresponds to the GenBank sequence UO8262. NMDAR1 receptor
It is a glutamate receptor involved in the processes underlying learning and memory. In addition, inhibition of the glutamate response by non-antagonistic (eg MK801 and dextrometallophane) and antagonistic (eg LY274614) NMDA receptor antagonists inhibits or reduces the development of morphine tolerance with long-term opiate administration. Many studies have shown this (Trujillo et al., Science, 251, 85-87, (1991); Elliott et al.
Pain, 56, 69-75 (1994); Wiesenfeld-Hallin, Z., Neuropsychopharm., 13, 34
7-56 (1995)). Early changes in the expression level of CLZ-34, which is highly homologous to the NMDA receptor, are of interest in terms of the ability of NMDA antagonists to block development of resistance to opiates.

Example 5 FIG. 19 shows AI415338: Soares mouse p3NMF19.5 Mus musculus cDNA clone IMAGE: 350746 3 ′, mRNA sequence; AI841003: UI-M-AM0-ado-e-04-0-UI.s1 NIH. -BAM
P-MAM Mus musculus cDNA clone UI-M-AM0-ado-e-04-0-UI 3 ', mRNA sequence; AI
413353: Soares mouse embryo NbME13.5 14.5 Mus musculus cDNA IMAGE: 356159
3 ', mRNA sequence; AI425991: Soares mouse embryo NbME13.5 14.5 Mus musculus c
DNA IMAGE: 426077 3 ′, mRNA sequence, which is a homologous sequence of the above four sequences arranged by computer (SEQ ID NO: 53).

Figure 20 shows EST AF006196: Mus musculus metalloprotease-disyntegrin MDC15 mRNA, complete cds (SEQ ID NO: 54). FIG. 21 shows C86593: Mus musculus fertilized egg cDNA 3′-terminal sequence, clone J0229.
E09 3 ', mRNA sequence; AI428410: Lifetech mouse embryo 13 5dpc 10666014 Mus
Muscrus cDNA clone IMAGE: 553802 3 ', mRNA sequence; AI561814: Stratagene mouse skin (# 937313) Mus Musculus cDNA clone IMAGE: 1227449 3', mR
The NA sequence and the homologous sequence of the above three sequences arranged by computer are shown (SEQ ID NO: 54).

Example 6 Characterization of CLZ 44 Male C57B1 / 6J mice (20-28 g) were housed as described above in Example 1.
The same experimental paradigm used in Example 1 for clozapine treatment is shown below.
Used for TOGA analysis. TOGA data shows that the 5'end is 6-carboxyfluorescein (6FAM
, ABI) labeled 5'-PCR primer (CGACGGTATCGGACGG; SEQ ID NO: 96) paired with a "universal"3'primer (SEQ ID NO: 23). PCR reaction products were electrophoretically separated on a 4.5% acrylamide gel and fluorescence data were obtained on an ABI 377 automated sequencer. The data was analyzed using GeneScan software (Perkin-Elmer).

As shown in Table 1, the TOGA analysis results indicate that CLZ-44 was slightly increased by clozapine treatment. Tables 2 and 3 show that CLZ-44 is an EST isolated from mouse kidney. To further characterize CLZ-44, control mice, clozapine-treated mice, haloperidol-treated mice, and ketanserin-treated mice to determine striatal / nuclear accumbence expression patterns 2 weeks after treatment. Northern blot analysis was performed (Fig. 22). Ketanserin is a 5HT _{2A / 2C} -specific antagonist and is used to determine the serotonergic involvement in the effects of these agents.

Briefly, agarose gel containing 2 μg of poly A-enhanced mRNA as well as size standards were electrophoresed on a 1.5% agarose gel containing formaldehyde, transferred to a biotrans membrane, and expressed in Expresshyb (Clonetech). Prehybridized for 30 minutes. CLZ-44 insert (25-100 ng) was labeled with [α- ³² P] -d CTP by oligonucleotide labeling with a specific activity of approximately 5 × 10 ⁸ cpm / μg, added to the prehybridization solution and incubated for 1 hour. did. Filters were washed at 68 ° C to extreme harshness (0.2 X SSC) (1 X SSC: 0.015 M NaCl and 0.0015 M sodium citrate) before applying 1 to Kodak X-AR film (Eastman Kodak, Rochester, NY). Exposed for up to a week. FIG. 22 illustrates the Northern blot analysis described above. As shown, after 2 weeks of treatment, CLZ-44 was increased by haloperidol and ketanserin but not by clozapine. This indicates that both dopamine D2 and 5HT2A / 2C receptors are involved in CLZ-44 expression regulation. The lack of effect of clozapine may indicate that antagonism of other receptors (ie 5HT ₃ , D4, D1) may abolish the effect of D2, 5HT ₂ receptors.

Example 7 Characterization of CLZ 38 Male C57B1 / 6J mice (20-28 g) were housed as described above in Example 1.
The same experimental paradigm used in Example 1 for clozapine treatment is shown below.
Used for TOGA analysis. TOGA data shows that the 5'end is 6-carboxyfluorescein (6FAM
, ABI) labeled 5'-PCR primer (CGACGGTATCGGTGCA; SEQ ID NO: 97) paired with a "universal"3'primer (SEQ ID NO: 23). PCR reaction products were electrophoretically separated on a 4.5% acrylamide gel and fluorescence data were obtained on an ABI 377 automated sequencer. The data was analyzed using GeneScan software (Perkin-Elmer).

Tables 2 and 3 show that CLZ-38 is an oligodendrocyte-specific protein mRNA. Further CLZ-38 characterization, Northern to determine striatum / nuclear accumence expression patterns of control mice and mice treated with clozapine for 45 minutes, 7 hours, 5 days, and 2 weeks. Blot analysis was performed (Figure 23).

Briefly, agarose gels containing 2 μg of poly A-enhanced mRNA as well as size standards were electrophoresed on a 1.5% agarose gel containing formaldehyde, transferred to a biotrans membrane and in Expresshyb (Clonetech). Prehybridized for 30 minutes. CLZ-38 insert (25-100 ng) was labeled with [α- ³² P] -d CTP by oligonucleotide labeling with a specific activity of about 5 × 10 ⁸ cpm / μg, added to the prehybridization solution and incubated for 1 hour. did. Filters were washed at 68 ° C to extreme harshness (0.2 X SSC) (1 X SSC: 0.015 M NaCl and 0.0015 M sodium citrate) before applying 1 to Kodak X-AR film (Eastman Kodak, Rochester, NY). Exposed for up to a week. FIG. 23 illustrates the Northern blot analysis described above. As shown, the pattern of CLZ-38 expression in clozapine-treated animals was similar to that observed in TOGA analysis.

Example 8 Characterization of CLZ 16 Male C57B1 / 6J mice (20-28 g) were housed as described above in Example 1.
The same experimental paradigm used in Example 1 for clozapine treatment is shown below.
Used for TOGA analysis. TOGA data shows that the 5'end is 6-carboxyfluorescein (6FAM
, ABI) labeled 5'-PCR primer (CGACGGTATCGGCTAG; SEQ ID NO: 97) paired with a "universal"3'primer (SEQ ID NO: 23). PCR reaction products were electrophoretically separated on a 4.5% acrylamide gel and fluorescence data were obtained on an ABI 377 automated sequencer. The data was analyzed using GeneScan software (Perkin-Elmer).

As shown in Table 1, the TOGA analysis results indicate that CLZ-16 is slightly reduced by clozapine treatment. Tables 2 and 3 show that CLZ-16 is an arm-repeat protein. Further characterization of CLZ-16, in situ hybridization analysis using an antisense cRNA probe oriented to the 3'end of CLZ-16, was performed on CLZ- in the anterior (24B) and posterior (24A) brains of mice. 16 mRN
This was done to show the pattern of A expression. Control mice and mice treated with 7.5 mg / kg clozapine were killed after 2 weeks. In situ hybridization was performed in the free-floating part (25 μM concentration). The parietal region is ³⁵ S-labeled with 10 ⁷ cpm / ml of CLZ-16, a single-stranded antisense cR.
Hybridized with NA probe at 55 ° C. for 16 hours.

The probe was derived from the 3'-terminated cDNA TOGA clone by Maxiscript Transcripti.
on Kit (Ambion, Austin, Tex.). Excess probe was added to 2X SSC containing 14 mM β-mercaptoethanol (1 X SSC = 0.015 M NaCl / 0.
[0015] 0.5 M NaCl / 0.05 M EDTA / 0.05 M Tris-H containing 4 μg / ml ribonuclease.
Incubated with Cl, ph 7.5 for 1 hour at 37 ° C. 55 X SSC / 50% formamide / 0.01 M β-mercaptoethanol under extremely severe conditions.
C. for 2 hours, then 0.1 X SSC / 0.01 M beta-mercaptoethanol / 0.
It was washed in 5% sarcosyl for 1 hour at 68 ° C. Slices were mounted on gelatin-coated slides and dehydrated with ethanol and chloroform prior to autoradiography. Slides were exposed to Kodak X-AR film for 1-4 days and then Ilford
Dipped in K-5 suspension. After 4 weeks, slides were developed on a Kodak D19 processor, fixed and counterstained with Richardson's blue stain.

As shown in Figures 24A and B, CLZ-16 mRNA is expressed throughout the mouse brain. FIG. 24A shows dark markers around the cortex and hippocampal formation and mild markers of the dorsal thalamus and posterior brain. Figure 24B shows a consistent label throughout.

Example 9 Characterization of CLZ-17 Male C57B1 / 6J mice (20-28 g) were housed as described above in Example 1.
The same experimental paradigm used in Example 1 for clozapine treatment is shown below.
Used for TOGA analysis. TOGA data shows that the 5'end is 6-carboxyfluorescein (6FAM
, ABI) labeled 5'-PCR primer (CGACGGTATCGGCTCA; SEQ ID NO: 99) paired with a "universal"3'primer (SEQ ID NO: 23). PCR reaction products were electrophoretically separated on a 4.5% acrylamide gel and fluorescence data were obtained on an ABI 377 automated sequencer. The data was analyzed using GeneScan software (Perkin-Elmer).

As shown in Table 1, the TOGA analysis results indicate that CLZ-17 is slightly reduced by clozapine treatment. Table 4 shows that CLZ-17 is compatible with some ESTs isolated from mouse tissue. Further characterization of CLZ-17, 3'of CLZ-17
In situ hybridization analysis using an antisense cRNA probe oriented toward the ends was performed using CLZ- in the anterior (25B) and posterior (25A) brains of mice.
17 was performed to show the pattern of mRNA expression.

In situ hybridization was performed on free-float from control mice and mice treated with 7.5 mg / kg clozapine for 2 weeks.
ing) portion (concentration of 25 μM). The top of the head is ³⁵ S at 10 ⁷ cpm / ml of CLZ-17
-Hybridized with a labeled, single-stranded antisense cRNA probe for 16 hours at 55 ° C. The probe was Maxiscript from a 3'-terminated cDNA TOGA clone.
Synthesized using the Transcription Kit (Ambion, Austin, Tex.). Excess probe was removed by washing as previously described in Example 8. Slices were mounted on gelatin-coated slides and dehydrated with ethanol and chloroform prior to autoradiography. Slides were exposed to Kodak X-AR film for 1-4 days and then placed in Ilford K-5 suspension. After 4 weeks, slides were developed on a Kodak D19 processor, fixed and counterstained with Richardson's blue stain.

[0370] Figure 25A-B shows an in situ hybridization analysis using an antisense cRNA probe oriented towards the 3'end of CLZ-17, parietal from posterior (25A) and anterior (25B) mouse brain. 3 shows the pattern of CLZ-17 mRNA expression in E. coli. As shown, CLZ-17 is expressed in cortex, hippocampus, striatum and amygdala.

Example 10 Characterization of CLZ 24 Male C57B1 / 6J mice (20-28 g) were housed as described above in Example 1.
The same experimental paradigm used in Example 1 for clozapine treatment is shown below.
Used for TOGA analysis. TOGA data shows that the 5'end is 6-carboxyfluorescein (6FAM
, ABI) and a 5'-PCR primer (CGACGGTATCGGGGCA; SEQ ID NO: 100) paired with a "universal"3'primer (SEQ ID NO: 23). PCR reaction products were electrophoretically separated on a 4.5% acrylamide gel and fluorescence data were obtained on an ABI 377 automated sequencer. The data was analyzed using GeneScan software (Perkin-Elmer).

As shown in Table 1, TOGA analysis results show that CLZ-24 is increased by clozapine treatment. Tables 2 and 3 show that CLZ-24 was isolated from rat tissue.
Indicates EST. Further characterization of CLZ-24, in situ hybridization analysis using an antisense cRNA probe oriented to the 3'end of CLZ-24, was performed in CLZ- in the anterior (26B) and posterior (26A) brains of mice. 24 was performed to show the pattern of mRNA expression.

In situ hybridization was performed using free-float from control mice and mice treated with 7.5 mg / kg clozapine for 2 weeks.
ing) portion (concentration of 25 μM). The top of the head is ³⁵ S at 10 ⁷ cpm / ml of CLZ-24
-Hybridized with a labeled, single-stranded antisense cRNA probe for 16 hours at 55 ° C. The probe was Maxiscript from a 3'-terminated cDNA TOGA clone.
Synthesized using the Transcription Kit (Ambion, Austin, Tex.). Excess probe was removed by washing as previously described in Example 8. Slices were mounted on gelatin-coated slides and dehydrated with ethanol and chloroform prior to autoradiography. Slides were exposed to Kodak X-AR film for 1-4 days and then placed in Ilford K-5 suspension. After 4 weeks, slides were developed on a Kodak D19 processor, fixed and counterstained with Richardson's blue stain.

[0374] Figures 26A-B show in situ hybridization analysis using an antisense cRNA probe oriented towards the 3'end of CLZ-24, from the parietal (26A) and brainstem as seen through the cerebral hemispheres of the mouse brain. The pattern of CLZ-24 mRNA expression at the crossing (26B) is shown. As shown, CLZ-24 mRNA is expressed throughout the cortex.

Example 11 Characterization of CLZ-26 Male C57B1 / 6J mice (20-28g) were housed as described above in Example 1.
The same experimental paradigm used in Example 1 for clozapine treatment is shown below.
Used for TOGA analysis. TOGA data shows that the 5'end is 6-carboxyfluorescein (6FAM
, ABI) and a 5'-PCR primer (CGACGGTATCGGGGCT; SEQ ID NO: 101) paired with a "universal"3'primer (SEQ ID NO: 23) labeled. PCR reaction products were electrophoretically separated on a 4.5% acrylamide gel and fluorescence data were obtained on an ABI 377 automated sequencer. The data was analyzed using GeneScan software (Perkin-Elmer).

As shown in Table 1, the TOGA analysis results indicate that CLZ-26 is slightly reduced by clozapine treatment. Table 4 shows that CLZ-26 is a metalloprotease-disyntegrin MDC15 mRNA. Further characterization of CLZ-26, of CLZ-26
In situ hybridization analysis using an antisense cRNA probe oriented to the 3'end was performed on CL in the anterior (27B) and posterior (27A) brains of mice.
This was done to show the pattern of Z-26 mRNA expression.

In situ hybridization was performed with the ³⁵ S-labeled single-stranded antisense cRNA probe of CLZ-26 along with the free-floating portion (
Concentration of 25 μM) as described in the previous example.

[0378] Figures 27A-B show in situ hybridization analysis using an antisense cRNA probe oriented towards the 3'end of CLZ-26, showing the head of the cerebral hemisphere at the level of hippocampal ridge formation in the mouse brain. Crest (27A) and cerebral hemisphere parietal in the order of striatum (2
7B) shows the pattern of CLZ-26 mRNA expression. As shown, CLZ-26 mRNA
Is expressed throughout the cortex.

Example 12 Characterization of CLZ 28 Male C57B1 / 6J mice (20-28 g) were housed as described above in Example 1.
The same experimental paradigm used in Example 1 for clozapine treatment is shown below.
Used for TOGA analysis. TOGA data shows that the 5'end is 6-carboxyfluorescein (6FAM
, ABI) and a 5'-PCR primer (CGACGGTATCGGGGTA; SEQ ID NO: 102) paired with a "universal"3'primer (SEQ ID NO: 23). PCR reaction products were electrophoretically separated on a 4.5% acrylamide gel and fluorescence data were obtained on an ABI 377 automated sequencer. The data was analyzed using GeneScan software (Perkin-Elmer).

As shown in Table 1, the TOGA analysis results indicate that CLZ-28 is reduced by clozapine treatment. Table 4 shows that CLZ-28 is compatible with some ESTs isolated from mouse tissues. Further characterization of CLZ-28, in situ hybridization analysis using an antisense cRNA probe directed to the 3'end of CLZ-28, was performed on CLZ- in the anterior (28B) and posterior (28A) brains of mice. 28 mRN
This was done to show the pattern of A expression.

In situ hybridization was performed with the ³⁵ S-labeled single-stranded antisense cRNA probe of CLZ-28 along with the free-floating portion (
Concentration of 25 μM) as described in the previous example.

Figures 28A-B show in situ hybridization analysis using an antisense cRNA probe oriented towards the 3'end of CLZ-28, from the cerebral hemisphere at the level of hippocampal ridge formation in mouse brain. The parietal region (28A) and the parietal region from the posterior part of the cerebral hemisphere (28B)
) Shows the pattern of CLZ-28 mRNA expression. As shown in Figures 28A and B, CLZ-28 mRNA is expressed throughout the cortex.

Example 13 Characterization of CLZ 3 Male C57B1 / 6J mice (20-28g) were housed as previously described in Example 1.
The same experimental paradigm used in Example 1 for clozapine treatment is shown below.
Used for TOGA analysis. TOGA data shows that the 5'end is 6-carboxyfluorescein (6FAM
, ABI) labeled 5'-PCR primer (CGACGGTATCGGAGTA; SEQ ID NO: 94) paired with a "universal"3'primer (SEQ ID NO: 23). PCR reaction products were electrophoretically separated on a 4.5% acrylamide gel and fluorescence data were obtained on an ABI 377 automated sequencer. The data was analyzed using GeneScan software (Perkin-Elmer).

As shown in Table 1, TOGA analysis results indicate that CLZ-3 is increased by clozapine treatment. Tables 2 and 3 show that CLZ-3 is a serine protease HTR.
Indicates that it is A mRNA. Further characterization of CLZ-3, in situ hybridization analysis using an antisense cRNA probe oriented towards the 3'end of CLZ-3, was performed on CLZ- in the anterior (29B) and posterior (29A) brains of mice. 3 was performed to show the pattern of mRNA expression.

In situ hybridization was performed with the ³⁵ S-labeled single-stranded antisense cRNA probe of CLZ-3 with the free-floating portion (2
5 μM concentration) and the method described in the previous example.

Figure 29A-B shows in situ hybridization analysis using an antisense cRNA probe oriented to the 3'end of CLZ-3, from the cerebral hemisphere at the level of hippocampal ridge formation in mouse brain. CLZ- at the parietal region (29A) and the intersection from the midbrain (29B)
3 shows the pattern of mRNA expression. CLZ-3 mR as shown in Figures 29A and B
NA is expressed in the cortex, thalamus, hippocampus, striatum and amygdala.

Example 14 Characterization of CLZ 34 Male C57B1 / 6J mice (20-28g) were housed as previously described in Example 1.
The same experimental paradigm used in Example 1 for clozapine treatment is shown below.
Used for TOGA analysis. TOGA data shows that the 5'end is 6-carboxyfluorescein (6FAM
, ABI) labeled "Universal"3'primer (SEQ ID NO: 23) and a 5'-PCR primer (CGACGGTATCGGTATT; SEQ ID NO: 103). PCR reaction products were electrophoretically separated on a 4.5% acrylamide gel and fluorescence data were obtained on an ABI 377 automated sequencer. The data was analyzed using GeneScan software (Perkin-Elmer).

As shown in Table 1, the TOGA analysis results indicate that CLZ-34 is increased by clozapine treatment. Tables 2 and 3 show that CLZ-34 is an N-methyl-D-aspartate receptor NMDAR1-2a subunit mRNA. Further characterization of CLZ-34, antisense cRNA oriented towards the 3'end of CLZ-34
In situ hybridization analysis using a probe was performed on the anterior brain (3
0B) and posterior brain (30A) to show the pattern of CLZ-34 mRNA expression.

In situ hybridization was performed with the ³⁵ S-labeled single-stranded antisense cRNA probe of CLZ-34 along with the free-floating portion (
Concentration of 25 μM) as described in the previous example.

[0390] Figures 30A-B show in situ hybridization analysis using an antisense cRNA probe oriented to the 3'end of CLZ-34, from the cerebral hemisphere at the level of hippocampal ridge formation in mouse brain. CLZ- at the parietal region (30A) and the intersection from the midbrain (30B)
34 shows the pattern of mRNA expression. CLZ-34 as shown in Figures 30A and B.
mRNA is expressed everywhere.

Example 15 Characterization of CLZ 43 Male C57B1 / 6J mice (20-28 g) were housed as previously described in Example 1.
The same experimental paradigm used in Example 1 for clozapine treatment is shown below.
Used for TOGA analysis. TOGA data shows that the 5'end is 6-carboxyfluorescein (6FAM
, ABI) labeled 5'-PCR primer (CGACGGTATCGGCTAA; SEQ ID NO: 104) paired with a "universal"3'primer (SEQ ID NO: 23). PCR reaction products were electrophoretically separated on a 4.5% acrylamide gel and fluorescence data were obtained on an ABI 377 automated sequencer. The data was analyzed using GeneScan software (Perkin-Elmer).

As shown in Table 1, TOGA analysis results indicate that CLZ-43 is increased by clozapine treatment. Tables 2 and 3 show that CLZ-43 is compatible with ESTs isolated from mouse tissues that are compatible with oxysterol binding protein family members. Further characterization of CLZ-43, in situ hybridization analysis using an antisense cRNA probe directed to the 3'end of CLZ-43, was performed in mice in the anterior brain (31C), midbrain (31A), and posterior CLZ-43 mR in the brain (31B)
This was done to show the pattern of NA expression.

In situ hybridization was performed with the ³⁵ S-labeled single-stranded antisense cRNA probe of CLZ-43 along with the free-floating portion (
Concentration of 25 μM) as described in the previous example.

FIG. 31A-C shows in situ hybridization analysis using an antisense cRNA probe oriented to the 3 ′ end of CLZ-43, showing intensity labeling in the cortex and striatum of the mouse brain. (31A-C) shows a pattern of parietal CLZ-43 mRNA expression in the cerebral hemisphere. Comparison with sections of brain obtained from control mice showed that CLZ-43 expression was increased approximately 10-fold by chronic treatment with clozapine (2 weeks).

Following cloning of mouse DST CLZ-43, BLAST analysis was performed. 5556 bp G
Human homology was identified as an enBank accession sequence (AB040884, also known as KIAA1451). An oligonucleotide was selected from this sequence, and the adult human brain
It was used to isolate the remaining 5'end of the human gene from the cDNA plasmid library. A 1717 bp cD that partially matches the human sequence using the method described below.
The NA clone (SEQ ID NO: 103) was isolated. This clone is GenBank accession sequence 5
'Providing an additional (new) 512 bp at the end. Sequence analysis shows that the position of the methionine start codon in the open reading frame is at base 562 of the 1717 bp clone (SEQ ID NO: 108). The open reading frame of the 1717 bp clone is a peptide of 385 amino acids (SEQ ID NO: 108,
Code 9).

The following method was used to isolate a 1717 bp cDNA clone. The target pool was a cDNA plasmid library made from adult human brain RNA. The oligonucleotide sequence used for hybridization was 5'-AAC AAG TCC G
It was TC CTG GCA TGG-3 '(SEQ ID NO: 88). The clone is the Gene Trapper kit (Li
It was isolated using the method specified by the manufacturer of fe Technologies). The capture oligonucleotide is biotin-1 using terminal deoxynucleotidyl transferase.
Prepared by end-modifying the oligonucleotide with 4-dCTP. cDNA
The plasmid pool was converted from double-stranded cDNA into single-stranded cDNA by the specific action of Gene II protein and exonuclease III. The single stranded cDNA pool was ligated with end-modified oligonucleotides to allow hybridization to occur for 30 minutes at room temperature. The reaction was subsequently mixed with magnetic beads coated with streptavidin. The single-stranded cDNA plasmid hybridized with the oligonucleotide was purified using a magnet to hold the magnetic beads in the reaction tube while all unbound components were washed away. Single-stranded plasmid DNA was dislodged from the oligonucleotide and repaired back to a double-stranded plasmid using a new sample of capture oligonucleotides and DNA polymerase. The repaired plasmid was transformed into bacteria and cultured on agar plates. The next day, bacterial colonies were independently picked and cultured overnight. Plasmid DNA was prepared from these small-preparations and used for sequence analysis.

22,000 b spanning 7 exons from homology match with human genome database
.p. more identified. Furthermore, CLZ-43 has been determined to be located on chromosome 12, which is not the chromosome traditionally associated with schizophrenia. The sequence data is for a protein with an open reading frame of 472 amino acids (SEQ ID NO: 1).
I made it clear that I coded 10). Comparisons with protein databases indicate that this protein is novel and is a member of a class of proteins that bind lipids, especially oxysterols.

Of the thousands of proteins expressed in the striatum, apoD and a novel oxysterol-binding protein are the few that are regulated by neuroleptic drugs, leading to schizophrenia in the brain. It reinforces the hypothesis that sterol homeostasis is a disease and, therefore, may have as many etiologies as atherosclerosis. The brain has much more cholesterol and 24 than any organ except the adrenal gland.
The extraordinary importance of the activity of the membranes of nerves and their myelinated cells with S-hydroxysterols is self-evident. The lipid bilayer of the membrane consists of glycerophospholipids and cholesterol, changes in its composition and saturation of the hydrocarbon chains determine the order and fluidity of the membrane. Such characteristics influence the binding of exogenous membrane proteins and, thus, the signaling of second messengers. As we have previously shown, a large proportion of highly enriched mRNAs in the striatum encode proteins that regulate second messenger signaling through the inner membrane. Therefore, disruption of the lipid metabolism system of whole nerves or whole organisms may first appear as a striatal disease. Solving the nature of the effects of neuroleptics on the properties of the membrane may attribute that issue to a greater focus.

Example 16 Characterization of CLZ-44 Male C57B1 / 6J mice (20-28 g) were housed as previously described in Example 1.
The same experimental paradigm used in Example 1 for clozapine treatment is shown below.
Used for TOGA analysis. TOGA data shows that the 5'end is 6-carboxyfluorescein (6FAM
, ABI) and a 5'-PCR primer (CGACGGTATCGGACGG; SEQ ID NO: 105) paired with a "universal"3'primer (SEQ ID NO: 23). PCR reaction products were electrophoretically separated on a 4.5% acrylamide gel and fluorescence data were obtained on an ABI 377 automated sequencer. The data was analyzed using GeneScan software (Perkin-Elmer).

As shown in Table 1, TOGA analysis results indicate that CLZ-44 is increased by clozapine treatment. Tables 2 and 3 show that CLZ-44 is compatible with ESTs isolated from mouse tissue. Further characterization of CLZ-44, in situ hybridization analysis using an antisense cRNA probe directed to the 3'end of CLZ-44, was performed in CLZ in the anterior (32A) and posterior (32B) brains of mice. -44 m
This was done to show the pattern of RNA expression.

In situ hybridization was performed with the ³⁵ S-labeled single-stranded antisense cRNA probe of CLZ-44 along with the free-floating portion (
Concentration of 25 μM) as described in the previous example.

FIG. 32A-B shows in situ hybridization analysis using an antisense cRNA probe oriented towards the 3 ′ end of CLZ-44 showing mouse hippocampus, hypothalamus, and temporal cortex ( 32A) shows the pattern of CLZ-44 mRNA expression in the parietal showing labeling and in the parietal showing cortical labeling (32B).

Example 17 Characterization of CLZ-64 Male C57B1 / 6J mice (20-28 g) were housed as described above in Example 1.
The same experimental paradigm used in Example 1 for clozapine treatment is shown below.
Used for TOGA analysis. TOGA data shows that the 5'end is 6-carboxyfluorescein (6FAM
, ABI) and a 5'-PCR primer (CGACGGTATCGGTCAT; SEQ ID NO: 106) paired with a "universal"3'primer (SEQ ID NO: 23). PCR reaction products were electrophoretically separated on a 4.5% acrylamide gel and fluorescence data were obtained on an ABI 377 automated sequencer. The data was analyzed using GeneScan software (Perkin-Elmer).

As shown in Table 1, the TOGA analysis results indicate that CLZ-64 is increased by clozapine treatment. Tables 2 and 3 show that CLZ-64 is compatible with ESTs isolated from mouse tissue and shares homology with mitochondrial enoyl-CoA hydratase mRNA. Further characterization of CLZ-64, in situ hybridization analysis using an antisense cRNA probe directed to the 3'end of CLZ-64, was performed in the anterior brain (33B) and midbrain (33A) of mice. This was done to show the pattern of 64 mRNA expression.

In situ hybridization was performed with the ³⁵ S-labeled single-stranded antisense cRNA probe of CLZ-64 along with the free-floating portion (
Concentration of 25 μM) as described in the previous example.

FIG. 33A-B shows in situ hybridization analysis using an antisense cRNA probe oriented to the 3 ′ end of CLZ-64, showing CLZ-64 mRNA at different parietal hemispheres in the mouse brain. Shows the pattern of expression of. As shown in Figures 33A and B, CLZ-64 is ubiquitously expressed.

These and other features, aspects, and advantages of the present invention will be further understood with reference to the following description, appended claims, and accompanying drawings.

[Brief description of drawings]

FIG. 1 is a schematic view of a 5 ′ PCR containing a parsing base AGTA.
It is a figure which shows the result of TOGA analysis using a primer, the following time, 7.5 m
mRN extracted from striatum / accumbens nucleus of mice treated with g / kg clozapine
Shows the PCR product produced from A: control (no clozapine), 45 minutes, 7 hours, 5 days, 12 days, and 14 days, where the vertical indicator line is present in the control sample and increased in the clozapine-treated sample. Showing a PCR product of about 106 base pairs.

2A-C show a more detailed analysis of the 106 base pair PCR product shown in FIG. The upper figure (FIG. 2A) shows a clone-specific primer (SEQ ID NO: 28) and a fluorescently labeled universal 3 ′ PCR primer (SEQ ID NO: 2).
The product produced in 3) is shown. FIG. 2B is a 5 ′ PCR primer, C-G-A-C.
-TOG of the prototype using -GGTATTAC-GGAGAGTAA (SEQ ID NO: 94) and fluorescently labeled universal 3'PCR primer (SEQ ID NO: 23)
The PCR product produced in the A reaction is shown. In the lower figure (Figure 2C), the transcripts from the upper and middle figures are overlaid, and the PCR product produced using the extended primers based on the cloned sequence is the same as the original PCR product. It has been shown to be length.

FIG. 3 is a diagram showing the results of TOGA analysis using a 5 ′ PCR primer containing a parsing base CACC, showing the striatum of a mouse treated with clozapine at 7.5 mg / kg for the following time. PCR products produced from mRNA extracted from the nucleus accumbens are shown: control (no clozapine), 45 minutes, 7 hours, 5 days, 1
For 2 and 14 days, the vertical indicator line indicates a PCR product of approximately 201 base pairs, present in the control sample and increased in the clozapine treated sample.

FIG. 4 shows Northern blot analysis of clone CLZ-5 (CACC201) showing poly A enriched mRNA from striatum / accumbens nucleus of mice treated with clozapine, Also, an agarose gel containing a size standard was blotted after electrophoresis, and radiolabeled CLZ-5 was used as a probe. Prior to mRNA extraction, mice were treated with clozapine (7.5 mg / kg) for the following times: control (no clozapine), 45 minutes, 7 hours, 5 days, 12 days, and 14 days.

FIG. 5 shows Northern blot analysis of clone CLZ-5 (CACC201) showing poly A from striatum / accumbens nucleus of mice treated with haloperidol.
Agarose gels containing RNA-enriched as well as size standards were blotted after electrophoresis using radiolabeled CLZ-5 as a probe. Prior to mRNA extraction, mice were treated with haloperidol (4 mg / kg) for the following times: control (no haloperidol), 45 minutes, 7 hours, 10 days, and 14 days.

6 is a TOGA analysis of the clone CLZ-5 shown in FIG. 3 and FIG.
It is a figure which compared the result of the northern blot analysis of the clone CLZ-5 shown by FIG.

7A-C are antisense cs targeting the 3'end of CLZ-5.
In situ hybridization analysis using RNA probe, CLZ-5mRN in mouse forebrain (7A), midbrain (7B), and hindbrain (7C).
The expression pattern of A is shown, wherein CLZ-5 is expressed in scattered glial cells and in the white matter area.

FIG. 8A-I is an in situ hybridization analysis using an antisense cRNA probe targeting the 3 ′ end of CLZ-5,
In the mouse forebrain (8A-C), midbrain (8D-F), and hindbrain (8G-I),
Saline-treated mice (upper row), mice treated with clozapine for 5 days (
CL in middle) and mice treated with clozapine for 14 days (lower)
Figure 5 shows the expression of Z-5 mRNA, where clozapine treatment induces expression in glial cells.

9A-H show control (9A-D) and clozapine treatment (9E-H).
) Animals, corpus callosum (cc, FIG. 9A, E); caudate putamen (C).
Shown are dark field micrographs of various brain sites including Pu, Figure 9B, F); anterior mating association (aca, Figure 9C, G); and pallidus (GP, Figure 9D, H).

10A-D show control (10A, C) and clozapine treatment (1).
Shown are darkfield micrographs of the internal (ic) (10A, B) and brightfield images of the optic tract (10C, D) from animals 0B, D).

11A-H show striae (11A, B, D, E) and visual observation of control saline (11A, B, C) and clozapine treated animals (11D, E, F). GFAP and apoD colocalization (co-loc) in the cord (11C, F).
where the thick arrows indicate GFAP and apoD mRNA.
Shows the co-localization of ApoD with the thin arrow indicating only the expression of apoD; 11G-H shows the anti-human apoD primary antibody (in the visual tract of control saline (11G) and clozapine treated animals (11H)). Novocastra, Newcastle, UK
3) shows the immunohistochemistry of apoD.

FIG. 12 shows Northern blot analysis of clone CLZ-5,
Agarose gels containing polyA-enriched mRNA from clozapine-treated cultured glial cells as well as size standards were blotted after electrophoresis and probed with radiolabeled CLZ-5. Cultured glial cells were treated with different concentrations of clozapine for different times prior to mRNA extraction as follows: A = control (no clozapine), B = 100 nM clozapine, 1 day, C = 1 μM clozapine, 1 day, D = 100 nM clozapine, 1 week, E = 1 μM clozapine, 1 week.

FIG. 13 is a diagram showing the result of TOGA analysis using a 5 ′ PCR primer containing a pershing base TTGT, and the results are as follows: 7.5 mg / kg
Shows PCR products produced from mRNA extracted from striatum / accumbens of mice treated with clozapine of: control (no clozapine), 45 minutes, 7 hours, 5 days, 12 days, and 14 days, Here, the vertical indicator line indicates a PCR product of about 266 base pairs that was down-regulated within 45 minutes in the clozapine-treated sample and remained down-regulated in the presence of clozapine for 14 days.

FIG. 14 is a diagram showing the result of TOGA analysis using a 5 ′ PCR primer containing a pershing base TTGT, which was produced from mRNA extracted from the brain of a mouse treated with morphine as follows. PCR products: control striatum (PS), acute treatment striatum (AS), withdrawal striatum (WS), control tonsils (PA),
Acute-treated tonsils (AA), chronically-treated tonsils (TA), and withdrawal tonsils (WA), where the vertical indicator line is more abundant in the control striatum than in the control tonsil and differs between striatum and tonsils by morphine Shows a PCR product of 266 base pairs regulated by.

FIG. 15 shows Northern blot analysis of clone CLZ-40 (TTGT266) showing poly A from striatum / accumbens nucleus of clozapine-treated mice.
Agarose gels containing RNA-enriched mRNA as well as size standards were blotted after electrophoresis using radiolabeled CLZ-40 as a probe. Mouse is mR
Prior to NA extraction, clozapine (7.5 mg / kg) was treated for the following times: control (no clozapine), 45 minutes, 7 hours, 5 days, 12 days, and 14 days.

16 is a graph comparing the results of TOGA analysis of clone CLZ-40 shown in FIG. 13 and Northern blot analysis of clone CLZ-40 shown in FIG.

FIG. 17A-B is an in situ hybridization analysis showing the expression of clone CLZ-40 mRNA in mouse brain using an antisense cRNA probe targeted to the 3 ′ end of CLZ-40, where 1
7A is nucleus accumbens (Acb) and pyriform cortex (P
ir) and 17B shows dentate gyrus (DG).

FIG. 18 is a diagram showing the results of TOGA analysis using a 5 ′ PCR primer containing a parsing base TATT, showing striatum / side of a mouse treated with 7.5 mg / kg of clozapine as follows. PCR products produced from mRNA extracted from nucleus accumbens; control (no clozapine), 45 minutes, 7 hours, 5 days, 12 days, and 14 days, where vertical indicator lines are present in control sample And shows a PCR product of about 89 base pairs that is differentially regulated by clozapine treatment time.

Figure 19 shows a consensus sequence from a cluster of 4 sequences: AI415388: Soares mouse p3NMF19.5 Mus musculus cDNA clone IMAGE:
350746 3 ', mRNA sequence; AI841003: UI-M-AM0-ado-e-04-0-UI.sl NIH- BMAP-MAM M
us musculus cDNA clone UI-M-AM0-ado-e-04-0-UI 3 ', mRNA sequence; AI413353:
Soares mouse embryo NbME 13.5 14.5 Mus musculus cDNA IMAGE: 356159 3 ', mRNA sequence; AI425991: Soares mouse embryo NbME 13.5 14.5 Mus musculus cDNA IMAGE; 4260
77 3 ', mRNA sequence.

FIG. 20 shows EST AF006196: Mus muscul.
us metalloprotease-disintegrin MDC15
The sequences of mRNA and complete cds are shown.

Figure 21 shows a consensus sequence from the following 3 sequence cluster: C86593: Mus musculus fertilized egg cDNA 3'end sequence, clone J0229E09 3 ', mR.
NA sequence; AI428410: Life Tech mouse embryo 13 5dpc 10666014 Mus musculus cDNA clone IMAGE: 553802 3 ', mRNA sequence; AI561814: Stratagene mouse skin (# 93731
3) Mus musculus cDNA clone IMAGE: 1227449 3 ', mRNA sequence.

FIG. 22 shows Northern blot analysis of clone CLZ - 44 (ACGG352) showing poly A from striatum / accumbens nucleus of clozapine treated mice.
Agarose gels containing RNA-enriched mRNA as well as size standards were blotted after electrophoresis using radiolabeled CLZ-44 as a probe. Mouse is mR
Before NA extraction, clozapine (7.5 mg / kg), haloperidol (4 mg / k)
g) or ketanserin (4 mg / kg) for 2 weeks.

FIG. 23 shows Northern blot analysis of clone CLZ-38 (TGCA109) showing poly A from striatum / accumbens nucleus of clozapine treated mice.
Agarose gels containing RNA-enriched mRNA as well as size standards were blotted after electrophoresis using radiolabeled CLZ-38 as a probe. Mouse is mR
Prior to NA extraction, clozapine (7.5 mg / kg) was treated for the following times: control (no clozapine), 45 minutes, 7 hours, 5 days, and 14 days.

FIG. 24A-B is an in situ hybridization analysis showing the expression pattern of clone CLZ-16 mRNA in a coronal section of mouse brain hemisphere using an antisense cRNA probe targeting the 3 ′ end of CLZ-16. Indicates. FIG. 24A shows that the cortex and hippocampal formation periphery are darkly labeled, and the thalamus dorsal and hindbrain are moderately labeled. In FIG. 24B, the whole is uniformly labeled.

25A-B are in situ hybridization analysis, using an antisense cRNA probe targeting the 3'end of CLZ-17, coronal section (25A) and midbrain of the hemisphere of mouse brain. The expression pattern of clone CLZ-17 mRNA in cross section (25B) is shown.

26A-B is an in situ hybridization analysis, using an antisense cRNA probe targeting the 3'end of CLZ-24, coronal section (26A) and brainstem section of hemisphere of mouse brain. (26B) shows the expression pattern of clone CLZ-24 mRNA in (26B).

27A-B are in situ hybridization analysis, using an antisense cRNA probe targeting the 3'end of CLZ-26, the level of hippocampal formation in the coronal section of the hemisphere of mouse brain ( 27A) and clone CLZ-26mRN at the striatal level (27B) in the coronal section of the hemisphere.
The expression pattern of A is shown.

28A-B is an in situ hybridization analysis using an antisense cRNA probe targeting the 3'end of CLZ-28 to detect hippocampal levels in coronal sections of mouse brain hemispheres (28A). ) And the expression pattern of clone CLZ-28 mRNA in the posterior coronal section (28B) of the brain hemisphere.

Figure 29A-B is an in situ hybridization analysis using an antisense cRNA probe targeting the 3'end of CLZ-3 to detect hippocampal levels in coronal sections of hemispheres of mouse brain (29A). ) And the cross section (29B) of the midbrain show the expression pattern of clone CLZ-3 mRNA.

FIG. 30A-B is an in situ hybridization analysis using an antisense cRNA probe targeting the 3 ′ end of CLZ-34 to detect hippocampal levels in coronal sections of hemispheres of mouse brain (30A). ) And the expression pattern of clone CLZ-34 mRNA in the cross section (30B) of the midbrain.

FIG. 31A-C is an in situ hybridization analysis, using an antisense cRNA probe targeted to the 3 ′ end of CLZ-43, labeled in the striatum of mouse brain (31A), in the cortex. Clone CLZ-43 in the coronal section of the hemisphere showing the marker (31B) and the strong marker in the striatum (31C).
The expression pattern of mRNA is shown.

32A-B are in situ hybridization analysis, labeling in the hippocampus, hypothalamus and temporal cortex of mouse brain with an antisense cRNA probe targeting the 3'end of CLZ-44. Coronal section showing (32A
), And clone CLZ-44m in coronal section (32B) showing cortical markers.
The expression pattern of RNA is shown.

FIG. 33A-B is an in situ hybridization analysis showing expression of clone CLZ-64 mRNA in different coronal sections of mouse brain hemispheres using an antisense cRNA probe targeted to the 3 ′ end of CLZ-64. The pattern is shown.

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C07K 14/47 C12N 1/15 4C085 16/18 1/19 4H045 C12N 1/15 1/21 1/19 C12P 21/08 1/21 C12Q 1/68 A 5/10 C12N 15/00 ZNAA C12P 21/08 5/00 A C12Q 1/68 A61K 37/02 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, K) , KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ , DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI , SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Satcliffe, Jay Gregore, California 92007, Cardiff-By-The-USA. — See, Via Timpo 2253 (72) Inventor Privile, Thomas M. California, USA 92109 San Diego, Pico Place 2609, Apartment Number 127 (72) Inventor Hillbush, Bryan, California 92130, San Diego, Willowmere Lane 5690 (72) Invention Hazel, Carl W. California, USA 92075, Soloana Beach, South Nard Avenue 247 F Term (Reference) 4B024 AA01 AA11 CA01 GA11 HA12 4B063 QA01 QA19 QQ01 QQ42 QQ79 QQ96 QR32 QR48 QR55 QS32 QX01 4B064 CC24 CA01 CA27 CA27 CA27 DA01 DA13 4B065 AA93Y AB01 AC14 BA02 CA24 CA25 CA44 CA46 4C084 AA01 AA02 AA06 AA07 BA01 BA22 CA53 DC50 ZA182 4C085 AA13 AA14 BB11 DD63 DD88 4H045 AA10 AA11 AA30 BA10 BA15 CA40 DA76 EA21 EA50 FA74 EA21 FA50 FA74

Claims (54)

[Claims] 1. SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, sequence No. 12, SEQ ID No. 13, Sequence No. 14, Sequence No. 15, Sequence No. 16, Sequence No. 17, Sequence No. 18, Sequence No. 19, Sequence No. 49, Sequence No. 50, Sequence No. 51, Sequence No. 52, Sequence No. 57 , SEQ ID NO: 58, SEQ ID NO: 5
9, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64
, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69,
An isolated nucleic acid molecule comprising a nucleotide selected from the group consisting of SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, and SEQ ID NO: 107. 2. SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, sequence No. 12, SEQ ID No. 13, Sequence No. 14, Sequence No. 15, Sequence No. 16, Sequence No. 17, Sequence No. 18, Sequence No. 19, Sequence No. 49, Sequence No. 50, Sequence No. 51, Sequence No. 52, Sequence No. 57 , SEQ ID NO: 58, SEQ ID NO: 5
9, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64
, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69,
An isolated polypeptide encoded by a nucleotide selected from the group consisting of SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, and SEQ ID NO: 107. 3. An isolated polypeptide of SEQ ID NO: 109. 4. An isolated polypeptide of SEQ ID NO: 110. 5. An isolated nucleic acid molecule comprising a polynucleotide that is at least 95% identical to the isolated nucleic acid molecule of claim 1. 6. An isolated nucleic acid molecule of at least 10 bases in length which hybridizes under stringent conditions with the isolated nucleic acid molecule of claim 1. 7. An isolated nucleic acid molecule encoding the polypeptide of claim 2. 8. An isolated nucleic acid molecule encoding a fragment of the polypeptide of claim 2. 9. An isolated nucleic acid molecule encoding a polypeptide antigenic determinant of the polypeptide of claim 2. 10. The polypeptide of claim 2, wherein the polypeptide has biological activity. 11. An isolated nucleic acid encoding a species homologue of the polypeptide of claim 2. 12. The isolated nucleic acid molecule of claim 1, wherein the nucleotide sequence comprises a contiguous nucleotide deletion from either the 5'or 3'ends. 13. A recombinant vector comprising the isolated nucleic acid molecule of claim 1. 14. A recombinant host cell containing the isolated nucleic acid molecule of claim 1. 15. The method for producing a recombinant host cell according to claim 14. 16. The recombinant host cell of claim 14, which comprises a vector sequence. 17. The isolated polypeptide of claim 2, wherein the isolated polypeptide comprises a contiguous amino acid deletion from either the C-terminus or the N-terminus. 18. An isolated antibody that specifically binds to the isolated polypeptide of claim 2. 19. An isolated antibody that specifically binds to the isolated polypeptide of claim 3. 20. An isolated antibody that specifically binds to the isolated polypeptide of claim 4. 21. The method according to claim 16, wherein the antibody is a monoclonal antibody.
19. The isolated antibody of claim 7 or claim 18. 22. The method according to claim 16 or 1, wherein the antibody is a polyclonal antibody.
19. The isolated antibody of claim 7 or claim 18. 23. A recombinant host cell expressing the isolated polypeptide of any one of claims 2, 3 or 4. 24. Produced by the following steps: (a) culturing the recombinant host cell of claim 14 under conditions such that the polypeptide is expressed, and (b) isolating the polypeptide. Isolated polypeptide. 25. A therapeutically effective amount of the polypeptide of any one of claims 2, 3, or 4 or the polynucleotide of claim 1 is administered to a mammalian patient. A method of preventing, treating, regulating, or ameliorating a medical condition comprising: 26. The medical condition is a neuropsychiatric disease.
The method described in. 27. Preventing a medical condition comprising administering to a mammalian patient a therapeutically effective amount of the antibody of any one of claims 18, 19 or 20. To treat, treat, regulate, or improve. 28. The medical condition is a neuropsychiatric disease.
The method described in. 29. A method for diagnosing a pathological condition of a patient and susceptibility to the pathological condition: (a) determining the presence or absence of a mutation of the polynucleotide according to claim 1, and (b) ) Diagnosing a pathological condition and susceptibility to the pathological condition based on the presence or absence of the aforementioned mutation. 30. The pathological condition is a neuropsychiatric disease.
9. The method according to 9. 30. An altered expression of a polypeptide encoded by the polynucleotide of claim 1, wherein the presence of the altered expression of the polypeptide is indicative of a pathological condition and susceptibility to the pathological condition. A method of diagnosing a pathological condition in a patient and susceptibility to the pathological condition, including detecting. 31. The method according to claim 30, wherein the change in expression is an increase in expression level or a decrease in expression level. 32. The pathological condition is a neuropsychiatric disease.
The method described in 0. 33. The method further comprises the steps of: obtaining a sample of the first organism from a patient suspected of having a neuropsychiatric disorder and a second sample from a suitable reference source; And a second sample to determine the amount of at least one polypeptide encoded by the polynucleotide of claim 1, and (b) comparing the amount of the polypeptide in the first and second samples. Including diagnosing the patient as having a neuropsychiatric disorder if the polypeptide of the first sample is in a greater or lesser amount than the polypeptide of the second sample 33. The method of claim 32. 34. The polynucleotide of claim 1, or any one of claims 2, 3, or 4 for the manufacture of a medicament for the treatment of a neuropsychiatric disorder. Use of the polypeptide according to paragraph. 35. Use of an antibody according to any one of claims 18, 19 or 20 for the manufacture of a medicament for the treatment of neuropsychiatric disorders. 36. For identifying a partner that binds to the polypeptide according to any one of claims 2, 3 and 4, (a) claim 2, claim 3 or claim. Contacting the polypeptide of any one of paragraphs 4 with a binding partner, and (b) determining whether the binding partner causes a change in the activity of the polypeptide.
A method that includes that. 37. A gene corresponding to the cDNA sequence of the isolated nucleic acid of claim 1. 38. A method of identifying the activity of a polypeptide expressed by a biological assay; (a) the polypeptide of any one of claims 2, 3 or 4 in a cell. (B) isolating the expressed polypeptide; (c) testing the activity of the expressed polypeptide by a biological assay; and (d) determining the activity of the expressed polypeptide based on the test results. Identifying. 39. Selected from the group consisting of DNA molecules shown in Table 1 having a 5'partial nucleotide sequence and length as described by digital address and having a regulatory pattern characteristic of neuroleptic drugs. , A substantially pure isolated DNA molecule suitable for use as a probe for a neuroleptic-regulated gene. 40. A mammalian protein encoded by a gene corresponding to the polynucleotide according to claim 1, or claim 2, claim 3, or claim 4.
35. A mammalian tissue sample comprising a quantity of a first antibody immunoreactive with a polypeptide encoded by the polynucleotide of any one of the claims, sufficient for at least one assay, and a suitable packaging material. A kit for detecting the presence of the polypeptide according to claim 2, 3, or 4. 41. The method of claim 40, further comprising a second antibody that binds to the first antibody.
The kit described in. 42. The kit according to claim 41, wherein the second antibody is labeled. 43. The kit of claim 42, wherein the label comprises an enzyme, isotope, fluorescent compound, colloidal metal, chemiluminescent compound, phosphorescent compound, or bioluminescent compound. 44. The polynucleotide of claim 1, or at least one.
A kit for detecting the presence of a gene encoding a protein, comprising an amount sufficient for at least one assay of a fragment thereof having 0 adjacent bases and a suitable packaging material. 45. To detect the presence of a nucleic acid encoding a protein in a sample of mammalian tissue; (a) sample the polynucleotide of claim 1 or a fragment thereof having at least 10 contiguous bases. Hybridizing with the nucleic acid of, and (b) detecting the presence of a hybridization product. 46. (a) Determining the presence or absence of a mutation in a polynucleotide of apolipoprotein D for diagnosing a neuropsychiatric disease or susceptibility to a neuropsychiatric disease in a patient, and (B) A method comprising diagnosing a neuropsychiatric disease or susceptibility to a neuropsychiatric disease based on the presence or absence of the aforementioned mutation. 47. Determining the presence or expression level of apolipoprotein D polypeptide in a biological sample for diagnosing a neuropsychiatric disorder or susceptibility to a neuropsychiatric disorder in a patient. And (b) diagnosing a neuropsychiatric disease or susceptibility to a neuropsychiatric disease based on the presence or expression level of an apolipoprotein D polypeptide. 48. The method of claim 46 or claim 47, wherein the neuropsychiatric disorder is schizophrenia. 49. The method of claim 46 or claim 47, wherein the neuropsychiatric disorder is a depressive disorder. 50. Determining the presence / absence of a mutation in the polynucleotide or polynucleotide fragment of SEQ ID NO: 2 for diagnosing a neuropsychiatric disease or susceptibility to a neuropsychiatric disease in a patient. And (b) diagnosing a neuropsychiatric disease or susceptibility to a neuropsychiatric disease based on the presence or absence of the aforementioned mutation. 51. For diagnosing a neuropsychiatric disease or susceptibility to a neuropsychiatric disease in a patient: (a) a translated polypeptide fragment of SEQ ID NO: 2 and an amino acid in a biological sample. Determining the presence or expression level of a polypeptide having at least 95% sequence identity, and (b) susceptibility to a neuropsychiatric disease or neuropsychiatric disease based on the presence or expression level of the polypeptide. Diagnosing, including methods. 52. The method of claim 50 or claim 51, wherein the neuropsychiatric disorder is schizophrenia. 53. The method of claim 50 or claim 51, wherein the neuropsychiatric disorder is a depressive disorder. 54. The neuropsychiatric disorder is a habit of drug addiction,
52. A method according to claim 50 or claim 51.