JP2002525039A - Neurotransmitter processing-related genes - Google Patents

Abstract

  (57) [Summary] The present invention provides five novel neurotransmitter processing-related genes and polypeptides encoded by those genes. The present invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. Further, the present invention provides a method for diagnosing, treating, or preventing a disease.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Some of the disclosures herein include those protected by copyright laws. The copyright holder shall not object to the reproduction of the patent or its disclosure in the case of a file or record in the Patent Office, but otherwise retains the copyright.

TECHNICAL FIELD The present invention relates to five genes involved in neurotransmitter processing and their corresponding gene products. The invention also relates to the use of these biomolecules in the prevention, diagnosis, treatment and evaluation of treatments for neurological and hormone related diseases, in particular Parkinson's disease, schizophrenia and epilepsy.

[0003] normal nerve function of the invention requires a precise balance of neurotransmitters. Disorders in balance can cause disease. For example, Parkinson's disease is triggered by an as yet unknown event that increases the sensitivity of dopaminergic neurons to oxidative processes leading to cell damage and cell death (Jenner, P. (1998) Mov. Disord. 1
3: 24-34). The resulting dopamine deficiency causes tremors and stiffness in patients with clinical symptoms of Parkinson's disease (Birkmayer, W. and P. Riedere
r (1986) Understandine the Neurotranmirters: Keys to the Workings of the Brain , Springer-Verlag, New York NY).

[0004] Many other diseases result from an improper balance between neurotransmitters. Central dopaminergic neurons have long been thought to play an important role in schizophrenia (Farde, L. (1997) Schizophr. Res. 28: 157-162),
There is an even more complex view that the interaction between dopamine, norepinephrine, and other neurotransmitters may be considered as a basis for disease (Carlsson,
A. et al. (1997) Life Sci. 61: 75-94). Dopamine and norepinephrine have been linked to hyperactive attention deficit disorder (Hechtman, L. (1994) J. Psychiary
Neuroaci. 19: 193-201). Norepinephrine has been linked to epilepsy in animal models (Szot, P. et al. (1996) Brain Res. Mol. Brain Res. 43: 233
-245: Clough, RW et al. (1998) Epilepsy Res. 29: 135-146); Janumpalli, S. et al. (1
998) J. Neurosci. 18: 2004-2008). Norepinephrine directly regulates gonadotropin-releasing hormone and may therefore be involved in reproductive disorders (Becu-Villalobos, D
(1997) Cell Mol. Neurobiol. 17: 699-715; Pau, KY. And HG.Spies (1997).
Chin. J. Physiol. 40: 181-196). Norepinephrine is also a dopamine β
-Regulates hydroxylase (DBH) and growth hormone. DBH inhibits growth in animal models (Malozowaki, S. et al. (1993) Acta Endocrinol. 12
9: 554-558).

[0005] In general, neurological disorders are treated with drugs that regulate neurotransmitter levels to maintain normal levels. Thus, any substrate, enzyme, receptor, or neurotransmitter transporter, as well as the genes involved in their regulation, have potential therapeutic uses. Disclosed herein are five new genes that show significant co-expression with genes known to be involved in the synthesis and release of the neurotransmitters dopamine, norepinephrine, epinephrine. These novel genes have been shown to prevent neurological and hormonal related diseases, specifically Parkinson's disease, schizophrenia, and epilepsy,
Useful for diagnosis, treatment, and evaluation of treatment.

[0006] In a first embodiment Summary of the Invention, the present invention provides a substantially purified containing one or more neurotransmitter processing specific genes and genes that are co-expressed in several biological samples A polynucleotide is provided. Each neurotransmitter processing specific gene is preferably selected from the following (a) to (d). (A) a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-5 (b)
A polynucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 6 (c) (a
A) a polynucleotide sequence complementary to the polynucleotide sequence of (b) or (d) a probe that hybridizes to the polynucleotide of (a), (b), or (c). And an expression vector comprising any of the above polynucleotides, and a host cell comprising the expression vector.

[0007] In a second embodiment, the present invention provides a substantially purified gene comprising a gene product of a gene that is co-expressed with one or more neurotransmitter processing-specific genes in a plurality of biological samples. A polypeptide is provided. Neurotransmitter processing specific genes include L-tyrosine hydroxylase (TH), aromatic amino acid decarboxylase (AADC), dopamine β-hydroxylase (DBH), nicotinic acetylcholine receptor α3 subunit precursor (nAchR-α3) , Secretogranin I and II, Rab3a, human cocaine and amphetamine-regulated transcript (hCART), vesicular monoamine transporter 1 (hVMAT1), and ARIX homeodomain proteins. Preferred embodiments include (a) SE
And a polypeptide sequence comprising at least six consecutive amino acids of the polypeptide sequence of (B) (a). Further, the present invention provides an antibody that specifically binds to any of the aforementioned polypeptides.

[0008] In another embodiment, the present invention provides a pharmaceutical composition comprising a polypeptide or polynucleotide of the present invention together with a suitable pharmaceutical carrier, and the use of such a composition for the treatment or prevention of said disease. A method for treating or preventing a disease comprising administering an effective amount to a patient in need of such treatment is provided.

In yet another embodiment, the invention provides a method for diagnosing a disease or condition associated with altered expression of a gene that is co-expressed with one or more neurotransmitter processing-specific genes in a sample. I will provide a. Here, each neurotransmitter processing specific gene is composed of L-tyrosine hydroxylase (TH), aromatic amino acid decarboxylase (AADC), dopamine β-hydroxylase (DBH), nicotinic acetylcholine receptor α3 subunit precursor ( nAchR-α3), secretogranins I and II, Rab3a, human cocaine and amphetamine-regulated transcripts (hCA
RT), vesicular monoamine transporter 1 (hVMAT1), and ARIX homeodomain proteins. The method includes providing a sample containing one or more co-expressed genes, and co-expressing the polynucleotide with the polynucleotide under conditions effective to purify the one or more hybridization complexes. A process of hybridizing the resulting gene and a process of detecting a hybridization complex, wherein the presence of one or more hybridization complexes has a correlation with the presence of a disease or disorder. Is included.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING The sequence listing provides an exemplary neurotransmitter processing-related gene sequence (SEQ ID NO: 1).
-5), and the polypeptide sequence obtained from the gene sequence of SEQ ID NO: 4 (SEQ ID NO: 4)
NO: 6). Each sequence is identified by a SEQ ID NO (SEQ ID NO :) and the Incyte clone number at which the sequence was first identified.

[0011] In embodiments herein for carrying out the invention, the notation of "an" and "the (this)" singular, unless it otherwise from the context it is clear, even multiple meanings Note that it includes. Thus, for example, reference to "a host cell" includes a plurality of such host cells, and reference to "antibody" includes reference to one or more antibodies and their equivalents well known to those of skill in the art. Represents.

Definitions Generally, "NSEQ" refers to a polynucleotide sequence of the present invention that includes SEQ ID NOs: 1-5. "PSEQ" refers to a polypeptide sequence of the invention that includes SEQ ID NO: 6.

“Variant” refers to either a polynucleotide or a polypeptide having a sequence branched from SEQ ID NO: 1-5 or SEQ ID NO: 6, respectively. Branching of a polynucleotide sequence results from alterations in variability, such as, for example, deletion, addition, and substitution of one or more nucleotides, which can also result from differences in codon usage. Each of these types of changes, alone or in combination with other changes, can occur one or more times in a given sequence. Polypeptide variants include sequences having at least one structural or functional characteristic of SEQ ID NO: 6.

“Fragment” refers to a nucleic acid sequence preferably having a length of at least 20 nucleic acids, more preferably a length of 40 nucleic acids, and most preferably a length of 60 nucleic acids, For example, a fragment consisting of nucleic acids 1-50 of SEQ ID NOs: 1-5 may be included. Also, a `` fragment '' is a polypeptide sequence preferably having a length of at least 5 to about 15 amino acids, most preferably at least 10 amino acids, for example,
It may also refer to a polypeptide sequence that retains some biological or immunological activity of SEQ ID NO: 6.

[0015] "Neurotransmitter-processing-spec
"ific genes)" refer to genes involved in processes such as synthesis or release of neurotransmitters. For the purposes of the present invention, the neurotransmitter is preferably dopamine, norepinephrine, epinephrine.

“Neurotransmitter processing-related gene” refers to a diagnosis, treatment, or prevention of a disease whose expression pattern is similar to that of a neurotransmitter processing-specific gene and is associated with a change in the expression of a neurotransmitter processing gene. New gene sequences useful for The gene sequences may also be useful in evaluating treatments for these diseases.

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

[0018] This invention is improper neurotransmitter processed into related diseases, neurological disorders and growth-related disorders in particular, and more as Parkinson's disease, neurological schizophrenia, and evaluation of therapy of epilepsy, prevention , Diagnostics, and therapeutics. These genes are described in U.S. Patent Application ("Prostate Cancer-Associated Genes", Walker
Et al., Serial No: 09 / 102,615 filed June 22, 1998), which is incorporated herein by reference.

The method first identifies polynucleotides that are expressed in a plurality of cDNA libraries. The polynucleotide may include a gene with a known function, a particular disease process, an intracellular compartment, a cell type, a tissue type, or a gene known to be specifically expressed in a species. Furthermore, polynucleotides include genes with unknown functions. The expression pattern of the known gene is then compared with the expression pattern of the unknown gene to determine whether it satisfies the specified threshold of coincidence probability. This comparison can identify a subset of polynucleotides that have a high probability of co-expression for a known gene. A high co-occurrence probability correlates with a specific co-occurrence probability threshold of less than 0.001, more preferably less than 0.00001.

Polynucleotides can be of various origins, including but not limited to eukaryotes such as humans, mice, rats, dogs, monkeys, plants, and yeasts, and prokaryotes such as bacteria and viruses. Obtained in a cDNA library derived from E. coli.
These nucleotides include, but are not limited to, expressed gene sequence fragments (ESTs), assembled polynucleotide sequences, and full-length gene coding regions, introns, regulatory sequences, 5 'untranslated regions, and 3' untranslated regions. A selection from various arrangements such as regions is possible. In order to obtain a statistically significant analysis, the polynucleotide must be expressed in at least three cDNA libraries. In one preferred embodiment, the polynucleotide is obtained from a sequence database such as the LIFESEQ database (Incyte Pharmaceuticals, Palo Alto CA).

The cDNA library used in the co-expression analysis of the present invention includes blood vessels, hearts,
Blood cells, cultured cells, connective tissue, epithelium, islets of Langerhans, neurons, phagocytic cells, bile duct, esophagus, gastrointestinal system, liver, pancreas, fetus, placenta, chromaffin system, endocrine glands, ovaries, uterus, penis, prostate, Seminal vesicle, testis, bone marrow, immune system, cartilage, muscle, skeleton,
Central nervous system, ganglia, glia, neuroendocrine system, peripheral nervous system, bronchi, larynx, lungs,
It may be obtained from the nose, pleura, ears, eyes, mouth, pharynx, exocrine glands, bladder, kidney, ureter and the like. The number of cDNA libraries selected can range from as little as 20 to over 10,000. The number of cDNA libraries is preferably 500 or more.

In a preferred embodiment, the gene sequences are assembled to reflect related sequences, such as assembling sequence fragments from a single transcript. Assembly of the polynucleotide sequence can be performed using various types of sequences such as, but not limited to, ESTs, extensions, or shotgun sequences. In a most preferred embodiment, the polynucleotide sequence is obtained from the US Provisional Patent Application (Lincoln et al., "Database and System fo
r Storing, Comparing and Displaying Related Biomolecular Sequence Inform
ation ", US Patent Application No. 60/079469, filed March 26, 1998), which is derived from human sequences assembled using the algorithm disclosed herein. Part of the book).

Experimentally, different expression of polynucleotides includes, but is not limited to, display of differences by spatial immobilization or gel electrophoresis, mismatch scanning of the genome,
It can be evaluated by a method including analysis of differences in expression, display of differences by transfer imaging, and the like. In addition, differential expression can be assessed by microarray technology. These methods can be used alone or in combination.

[0024] Neurotransmitter processing-specific genes are selected based on the current use of these genes as therapeutic targets for diseases associated with incorrect neurotransmitter processing or as diagnostic markers. obtain. Neurotransmission processing-related genes include L-tyrosine hydroxylase (TH), aromatic amino acid decarboxylase (AADC), dopamine β-hydroxylase (DBH), nicotinic acetylcholine receptor α3 subunit precursor (nAchR-α3), Secretogranin I and
II, Rab3a, human cocaine and amphetamine-regulated transcript (hCART), vesicular monoamine transporter 1 (hVMAT1), and ARIX homeodomain proteins.

The procedure for identifying new genes that exhibit a statistically significant co-expression pattern with known neurotransmitter processing-related genes is as follows. First, the cD
Confirm the presence or absence of the gene sequence in the NA library. If at least one cDNA fragment corresponding to the gene is detected in a cDNA sample from the library, the gene is present in the cDNA library and the corresponding cDNA
If no fragment is detected in the sample, the gene is absent.

Second, the significance of co-expression of genes is assessed using a probabilistic method that measures the probability of coincidence of co-expression. The probabilistic method Fisher's exact test (Fisher exact test), may be chi ² test, or κ-test, and the like. These tests and their applications are well known to those skilled in the art and can be found in standard statistical texts (
Agresti, A. (1990) Categorical Data Analysis.Wiley, New York, NY; Rice,
JA (1988) Mathematical Statistics and Data Analysis.Wadsworth & Bro
oks / Cole, Pacific Grove, CA). Bonferroni correction (Rice, supra , page 384) to correct for statistical results of multiple other genes for a single gene.
Can be used in combination with one of the stochastic methods. In a preferred embodiment, the probability of a coincidence is measured by Fisher's exact test, and the threshold for the probability of a coincidence is set to less than 0.001, more preferably less than 0.00001.

To determine whether the two genes A and B have similar co-expression patterns, a vector of developmental data as shown in Table 1 can be created (where the gene The presence is represented by 1 and the absence is represented by 0). 0 indicates that the gene did not occur in the library, 1 indicates that the gene occurred at least once.

[0028]

[Table 1]

For a given pair of genes, the occurrence data in Table 1 can be summarized in a 2 × 2 contingency table.

[0030]

[Table 2]

Table 2 shows coincidence data for gene A and gene B in a total of 30 libraries. In the library, gene A and gene B each occur 10 times. Table 2 shows the following items: (1) Gene A and Gene B
(2) Number of times that both gene A and gene B are not present in the library, (3) Number of times that gene A is present and gene B is not present, (4) Gene
The number of times B is present and gene A is not present is summarized. The top left item is the number of times two genes occurred simultaneously in the library, and the middle right item is the number of times no genes occurred in the library. The other diagonal items are the number of times one gene occurred and the other gene did not. Gene A and gene B were both present eight times, both were absent 18 times, gene A was present and gene B was not present twice, and gene B was present Gene A
Was not present twice. The probability (P-value) that the above correlation occurs by chance coincidence, calculated using Fisher's exact test, is 0.0003. The correlation is usually considered significant when the P value is less than 0.01 (Agresti, supra; Ri
ce, supra ).

This method of assessing the probability of co-expression of two genes is based on several assumptions. This method assumes that the libraries are sampled independently and similarly. However, in practice, the selected cDNA libraries are not completely independent, as more than one library may have been obtained from one patient or one tissue. Also, since a different number of cDNAs (typically 5,000 to 10,000 per library) can be sequenced from each library, they are not sampled exactly the same. In addition, since the co-expression probability by Fisher's exact test is calculated for each gene for 41,419 other genes, many statistics are required by requiring a more exact P value than the standard P value of 0.01. The probability is corrected for the statistical test.

Using the co-expression analysis method, five new genes were identified that showed strong association or co-expression with known neurotransmitter processing-related genes. Table 5 to Table 10
Indicate that the expression of the five novel genes has a direct or indirect relationship with the expression of known neurotransmitter processing-related genes. Therefore,
The novel gene is used to diagnose neurotransmitter processing-related diseases such as Parkinson's disease, schizophrenia, epilepsy, female reproductive disorders, and attention deficit disorders,
It can be used in the treatment or prevention, or in evaluating the treatment of those diseases.
In addition, the gene products of the five novel genes are potential therapeutic proteins and therapeutic targets for those diseases.

Thus, in one embodiment, the invention encompasses a polynucleotide sequence comprising the sequence of SEQ ID NO: 1-5. These five polynucleotides have been shown to have a strong co-expression relationship with known neurotransmitter processing-related genes by the methods of the present invention. Also, the present invention provides a variant of the polynucleotide sequence,
Includes the complementary sequence, or 18 contiguous nucleotides in the above sequence. Generally, a variant polynucleotide sequence has at least about 70%, more preferably at least about 85%, and most preferably at least about 95% polynucleotide sequence identity to the NSEQ.

One of the preferred methods for identifying variants using NSEQ and / or PSEQ sequences is to use primates (pri), rodents (rod), and mammals (mam), vertebrates (Gen) in GenBank. vrtp) and eukaryote (eukp) databases, SwissProt, BLOCKS (Bairoch,
A. et al. (1997) Nucleic Acids Res. 25: 217-221), PFAM, and other databases containing previously identified annotated motifs, sequences, and gene functions. BLAST (B), along with a method for searching for primary sequence patterns with gap penalties for secondary structure (Smith, T. et al.
asic Local Alignment Search Tool; Altschul, SF (1993) J. Mol. Evol 36:
290-300; and Altschul et al. (1990) J. Mol. Biol. 215: 403-410), BLOCKS (Heni
koff S. and Henikoff GJ (1991) Nucleic Acids Research 19: 6565-6572),
Hidden Markov Model (HMM; Eddy, SR. (1996) Cur. Opin. Str. Biol. 6: 361-36
5; and Sonnhammer, ELL et al. (1997) Proteins 28: 405-420) and the like to manipulate and analyze nucleotide and amino acid sequences. These databases, algorithms, and other methods are well known to those skilled in the art and are described in Ausubel, FM et al. (1997; Short Protocols in Molecular Biolocy , John Wi
ley & Sons, New York, NY) and Meyers, RA (1995; Molecular Biology and Biotechnology , Wiley VCH, Inc, New York, NY, p 856-853).

Also included within the scope of the present invention are stringent sequences under stringent conditions.
There are polynucleotide sequences that can hybridize with ID NOs: 1-5, and fragments thereof. Stringent conditions can be defined by salt concentration, temperature, and other chemicals, as well as conditions well known to those skilled in the art.

For example, stringent salt concentrations are usually less than about 750 mM NaCl and less than 75 mM trisodium citrate, preferably less than about 500 mM NaCl and less than 50 mM trisodium citrate, most preferably less than about 250 mM. NaCl and less than 25 mM trisodium citrate. Stringent temperature conditions are usually at least about 30 ° C,
More preferably, the temperature is at least about 37 ° C, most preferably at least about 42 ° C. It is well known to those skilled in the art to change additional parameters such as, for example, hybridization time, concentration of detergent (sodium dodecyl sulfate (SDS)) or solvent (formamide), and the presence or absence of carrier DNA. Further modifications to these conditions will be readily apparent to those skilled in the art (Wahl, GM and SL Berger (1
987) Methods Enzymol. 152: 399-407; Kimmel, AR (1987) Methods Enzymol.
152: 507-511; Ausubel, supra ; Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Press, Plainview, NY).

[0038] The polynucleotide sequence encoding NSEQ or PSEQ may be extended utilizing the partial nucleotide sequence using various PCR-based methods well known to those skilled in the art, such as promoters and regulatory elements. It is possible to detect upstream sequences (eg, Dieffenbach, CW and GS Dveksler (1995; PCR Primer, a La
boratory Manual, Cold Spring Harbor Press, Plainview, NY, pp.1-5; Sarkar
, G. (1993; PCR Methods Applic. 2: 318-322); Triglia, T. et al. (1988; Nucleic
Acids Res. 16: 8186); Lagerstrom, M. et al. (1991; PCR Methods Applic. 1: 111-
119); and Parker, JD et al. (1991; Nucleic Acids Res. 19: 3055-306))
. Furthermore, walking in genomic DNA can be performed using PCR and nested primers. This method eliminates the need to screen libraries and is useful for finding intron / exon junctions. For all PCR-based methods, commercially available software such as OLIGO 4.06 software (National Biosciences, Plymouth MN) or other suitable programs can be used to reduce the length of 18-30 nucleotides, GC content. Primers can be designed to anneal to the template at a temperature of 50% or more and about 68-72 ° C.

[0039] In another embodiment of the present invention, the polynucleotide sequence encoding NSEQ or PSEQ is cloned into a recombinant DNA molecule to provide a P in a suitable host cell.
It can direct the expression of a polypeptide encoded by SEQ or NSEQ, or a structural or functional fragment thereof. Due to the inherent degeneracy of the genetic code, other DNA sequences encoding substantially the same or a functionally equivalent amino acid sequence may be generated, which express the polypeptide encoded by the PSEQ or NSEQ Can be used to The nucleotide sequences of the present invention can be recombined using methods well known to those skilled in the art to modify the nucleotide sequence for various purposes. The purpose includes, but is not limited to, cloning, processing, and altering expression of the gene product. Nucleotide sequences can be manipulated using DNA shuffling by random fragmentation and PCR reconstruction of gene fragments and synthetic oligonucleotides. For example, oligonucleotide-mediated site-directed mutagenesis can be used to introduce mutations that create new restriction sites, alter glycosylation patterns, alter codon preferences, generate splice variants, etc. .

To express a biologically active polypeptide encoded by NSEQ, a polynucleotide sequence encoding NSEQ or PSEQ, or a derivative thereof, is inserted into a suitable vector (ie, inserted into a suitable host). (A vector containing sequences necessary for the regulation of transcription and translation of the coding sequence). These elements include, for example, enhancers, constitutive and inducible promoters, and 5 'and 3' untranslated regions in vectors and in polynucleotide sequences encoding NSEQ or PSEQ. Using methods well known to those skilled in the art,
Expression vectors can be made that contain a polynucleotide sequence encoding NSEQ or PSEQ, and appropriate transcriptional and translational regulatory regions. These methods, recombinant DNA technology in the in vit ro, include genetic recombination or the like in the synthetic techniques, and in vivo (
See, for example, Sambrook, supra, and Ausuhel, supra).

A variety of expression vector / host cell systems can be used to introduce and express a polynucleotide sequence encoding NSEQ or PSEQ. These include, but are not limited to, microorganisms such as bacteria transformed with a recombinant bacteriophage, plasmid, or cosmid DNA expression vector, yeast transformed with a yeast expression vector, and viral expression vectors ( Baculovirus) or a plant cell line transformed with a virus expression vector, cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV), or a bacterial expression vector (Ti or pBR322 plasmid). Or animal cell lines. The invention is not limited by the host cell used. For long-term production of recombinant proteins in mammalian systems, stable expression of the polypeptide encoded by NSEQ in a cell line is desirable. For example, a sequence encoding NSEQ or PSEQ can be transformed into a cell line using an expression vector that can contain the viral origin of replication, an endogenous expression element and a selectable marker gene on the same or separate vectors. .

In general, host cells that express a PSEQ, including an NSEQ, can be identified by various methods well known to those skilled in the art. These methods include, but are not limited to, DNA
-Includes protein bioassay or immunoassay techniques, including membrane, solution, or chip-based techniques, for performing DNA or DNA-RNA hybridization, PCR amplification, and nucleic acid or protein sequence detection and quantification. Immunological methods for detecting and measuring PSEQ expression using either specific polyclonal or monoclonal antibodies are well known to those of skill in the art. Examples of such techniques include enzyme-linked immunoassay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorter (FACS).

A host cell transformed with a polynucleotide sequence encoding NSEQ or PSEQ
It can be cultured under conditions suitable for expressing the protein and recovering it from the medium. Proteins produced by the transformed cells can be secreted or retained intracellularly depending on the sequence and vector used. As will be appreciated by those skilled in the art, an expression vector comprising a polynucleotide of an NSEQ or a polynucleotide encoding a PSEQ can be transferred through the plasma membrane of a prokaryotic or eukaryotic cell to the PSEQ or
It can be designed to include a signal sequence that directs secretion of the polypeptide encoded by NSEQ.

In addition, a host cell strain may be chosen for its ability to process proteins expressed in the desired form or to regulate the expression of the inserted sequences. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, acylation, and the like. Post-translational processing that cuts off the "prepro" portion of the protein can be used to identify protein targeting, folding, and / or activity. A variety of host cells (eg, CHO, HeLa, MDCK, HEK293, and WI38) with specific cellular devices and characteristic mechanisms for post-translational activity are available from the American Type Culture Collectio.
n (ATCC, Bethesda, MD) and can be selected to ensure the correct modification and processing of the foreign protein.

In another embodiment of the present invention, a natural, modified or recombinant NSEQ or PS
The nucleic acid sequence encoding the EQ is ligated to the heterologous sequence such that translation of the fusion protein comprising the heterologous protein portion occurs in any of the aforementioned host systems. Such heterologous protein moieties facilitate purification of the fusion protein using a commercially available affinity matrix. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP
), Sialedoxin (Trx), calmodulin-binding peptide (CBP), 6-His, FLA
G, c-myc, hemagglutinin (HA), and epitopes of monoclonal antibodies.

In another embodiment, all or part of the sequence encoding NSEQ or PSEQ is synthesized using chemical methods well known to those of skill in the art (eg, Caruthers, MH et al. (1980) N
ucl. Acids Symp. Ser. (7): 215-223; Hom, T. et al. (1980) Nucl. Acids Symp. Se.
r. (7): 225-232; and Ausubel, supra ). Alternatively, the polypeptide sequence itself, encoded by PSEQ or NSEQ, or a fragment thereof, can be synthesized using chemical methods. For example, peptide synthesis can be performed using various solid-phase techniques (
Roberge, JY et al. (1995) Science 269: 202-204). ABI 431 A Peptide Sy
Synthesis can be automated using nthesizer (PE Biosystems, Foster City CA). Further, the amino acid sequence encoded by the PSEQ or NSEQ, or any portion thereof, may be modified during direct synthesis and / or linked to another protein or any portion thereof to form Peptide variants can be generated.

In another embodiment, the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 6, or a fragment thereof.

Diagnosis and Treatment The genes of the present invention are useful for diagnosing, preventing and treating diseases associated with neurotransmitter processing, in particular neurological and growth-related diseases, more particularly Parkinson's disease, schizophrenia and epilepsy. , And in the evaluation of treatment. Furthermore, the amino acid sequences encoded by this novel gene can be therapeutic proteins and therapeutic targets for these diseases.

In a preferred embodiment, the polynucleotide sequence of the polynucleotide encoded by NSEQ or PSEQ is used for diagnostic purposes to determine the absence, presence, and overexpression of PSEQ, The regulation of the level of the polypeptide encoded by the mRNA or NSEQ is monitored. The polynucleotide is at least 18 nucleotides in length, and may comprise complementary RNA and DNA molecules, branched nucleic acids,
And peptide nucleic acids (PNA). Alternatively, the polynucleotide is used to detect and quantify gene expression in a sample in which expression of the polypeptide encoded by SEQ or NSEQ correlates with disease. In addition, polynucleotides encoding NSEQ or PSEQ can be used to detect polymorphisms in disease-related genes. These polymorphisms can be detected at the transcript cDNA or genomic level.

The specificity of the probe, ie, the region in which the probe is highly specific (eg,
Whether it was made from a 5 'regulatory region) or a less specific region (eg, a conserved motif), and the rigor (maximum, high, medium, or low) of hybridization or amplification The nature of the probe
It is determined whether only the sequence encoding PSEQ is identified, or whether it also identifies allelic variants and related sequences.

[0051] Probes are also used to detect related sequences and preferably have at least 50% sequence identity with either the NSEQ or PSEQ encoding sequence.

[0052] Means for producing a hybridization probe specific for DNA encoding PSEQ include methods in which a polynucleotide sequence encoding NSEQ or PSEQ is cloned into a vector to produce an mRNA probe. included. Such vectors are well known to those of skill in the art and are commercially available, and can be used for the synthesis of RNA probes in vitro by adding an appropriate RNA polymerase and appropriate labeled nucleotides. . Hybridization probes can be labeled with various reporter molecules (eg, radionuclides such as ³² P or ³⁵ S), or enzyme labels such as alkaline phosphatase, fluorescent labels, etc., that bind to the probe via an avidin / biotin binding system. Can be done. Polynucleotide sequences encoding PSEQ can be used in Southern or Northern blots, dot blots, or other membrane-based techniques using tissue or body fluids of patients, PCR techniques, or microarrays to express PSEQ expression. Changes can be detected. Such qualitative or quantitative methods are well known to those skilled in the art.

The nucleotide sequence encoding NSEQ or PSEQ is labeled by standard methods,
It can be added to a body fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After an appropriate incubation time, the sample is washed and the signal is quantified and compared to a standard value. If the amount of signal in the patient's sample is significantly different from the standard value, a change in the concentration of the nucleotide sequence encoding NSEQ and PSEQ in the sample indicates the presence of the associated disease. Such assays can also be used to evaluate the efficacy of a particular therapeutic treatment in animal studies, clinical trials, or for monitoring the treatment of individual patients.

[0054] Once the presence of the disease is confirmed and the treatment protocol is initiated, the hybridization or amplification assay is repeated regularly to determine whether expression levels in the patient have begun to approach those observed in normal subjects. Can be determined. Results from continuous assays are used to show the efficacy of treatment over a period ranging from days to months.

The polynucleotide can be used in the diagnosis of various diseases related to neurotransmitter processing, such as neurological diseases. Such diseases include, but are not limited to, for example, immobility, Alzheimer's disease, amnesia, amyotrophic lateral sclerosis,
Bipolar disorder, catatonia, cerebral tumor, dementia, depression, diabetic neuropathy, Down syndrome, extrapyramidal terminal deficiency syndrome, dystonia, epilepsy, Huntington's disease,
Peripheral nerve diseases, multiple sclerosis, neuropyramidosis, Parkinson's disease, delusional psychosis, postherpetic neuralgia, schizophrenia, and Tourette's disease, as well as abnormal prolactin production, fallopian tube, and ovulation Abnormalities, endometriosis, disrupted estrous cycle, disrupted menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, endometrial or ovarian tumors, uterine fibroids, autoimmune abnormalities, ectopic pregnancy, and malformations Includes development, breast, testicular, and prostate cancer, mammary fibrocystic disease, hypertrophy, disrupted spermatogenesis, abnormal sperm physiology, prostatitis, Peyronie's disease, and reproductive disorders such as gynecomastia.

Alternatively, the polynucleotide can be used as a target in a microarray. Microarrays can be used to simultaneously monitor the expression levels of many genes and identify splice variants, mutations, and polymorphisms. This information can be used to determine gene function, understand the genetic basis of disease, diagnose disease,
And for the development of therapeutic agents and monitoring of their effects.

In yet another embodiment, the polynucleotides can be used to generate hybridization probes useful for mapping sequences of naturally occurring genomes. Fluorescence in situ hybridization (FISH) can be correlated with other physical chromosome mapping techniques and genetic map data (eg, Heinz-Ulrich, et al.
995) in Meyers, supra , pp. 965-968).

In another embodiment, antibodies that specifically bind to PSEQ can be used in the diagnosis of a disease characterized by over or under expression of the polypeptide encoded by PSEQ or NSEQ. Alternatively, competitive drug screening assays can be used in which neutralizing antibodies capable of binding to the polypeptide encoded by the PSEQ or NSEQ compete with the test compound for binding to the polypeptide. In this method, antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with a polypeptide encoded by a PSEQ or NSEQ.
Diagnostic assays for polypeptides encoded by PSEQ or NSEQ utilize antibodies and labels to detect PSEQ or NSEQ in human body fluids or extracts of cells or tissues.
And methods for detecting the polypeptide encoded by. Various protocols for measuring polypeptides encoded by PSEQ or NSEQ, including ELISA, RIA, and FACS, are well known to those of skill in the art, and are used to measure the expression level of a polypeptide encoded by PSEQ or NSEQ. Provide the basis for diagnosing change or abnormality. The normal or standard value of PSEQ expression can be determined by comparing PSEQ or NS with a fluid or cell extract taken from a normal subject (preferably human) under conditions suitable for complex formation.
It is determined by binding to the polypeptide encoded by the EQ.
The standard complex formation can be quantified by various methods (preferably photometric means). The amount of PSEQ or NSEQ encoded polypeptide expressed in subject, control, and disease specimens from biopsy tissue is compared to a standard value.
The deviation between the standard value and the subject's value establishes parameters for diagnosing or monitoring the disease.

In another embodiment, the polynucleotides and polypeptides of the present invention can be used to treat or monitor therapeutic treatment of the specific diseases described above. NS
An EQ polynucleotide or a polynucleotide encoding a PSEQ, or any fragment or complementary molecule thereof, can be used for therapeutic purposes. In one embodiment, molecules complementary to the polynucleotide of NSEQ or the polynucleotide encoding PSEQ can be used in situations where it is desirable to prevent transcription or translation of mRNA.

Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or expression vectors derived from various bacterial plasmids, can be used to deliver nucleotide sequences to targeted organs, tissues, or cell populations. Can be used. Vectors expressing nucleic acid sequences complementary to a polynucleotide encoding a PSEQ can be made using methods well known to those of skill in the art (eg, Sambro).
ok, supra ; and Ausubel, supra ).

A gene having a polynucleotide sequence encoding NSEQ or PSEQ can be produced by transforming a cell or tissue with a vector that expresses a polynucleotide or fragment thereof encoding PSEQ at high levels. Such constructs can be used to introduce non-translatable sense or antisense sequences into cells. Oligonucleotides derived from the transcription start site (e.g., a region generally in the range of -10 to +10 of the start site) are preferred. Similarly, inhibition can be achieved using triple helix base pairing. Triple helix pairing is useful because the triple helix impairs the ability to open enough double helices for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triple helix DNA have been disclosed in the literature (see, for example, Huber, BE and BI. Carr, Molecular and Im munolocic Anoroaches , Futura Publishing Co., Mt. Kisco NY, pp. 163-177). Gee, JE. Et al. (1994)). In addition, specific cleavage of RNA can be catalyzed using a ribozyme which is an enzymatic RNA molecule.

[0062] RNA molecules can be modified to increase intracellular stability or half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5 'and / or 3' end of the molecule, and the addition of phosphorothionate or 2'O-methyl instead of a phosphodiesterase linkage within the backbone of the molecule. Use is included. This idea is inherent in the production of PNAs and can be extended to all of these molecules by including untranslated bases. The non-translatable bases include
Adenine, cytidine, guanine, thymine and uridine are not readily recognized by endogenous endonucleases, as well as acetyl-, methyl-, thio-, and similar modified forms, as well as inosine, queosine, and wybutosin.
ine).

A number of methods are available for introducing vectors into cells or tissues,
They are also suitable for in vivo , in vitro and ex vivo use. In the case of ex vivo treatment, the vector is introduced into stem cells taken from a patient and propagated as a clone for autotransplantation back to the same patient. Also, transfection, liposome injection,
Alternatively, delivery with a polycationic amino polymer can be performed using methods well known to those skilled in the art (eg, Goldman, CK et al. (1997) Nature Biotechnology 15: 4).
62-466).

Further, an antagonist or antibody of the polypeptide encoded by PSEQ or NSEQ is administered to the patient for the treatment or prevention of a disease associated with the synthesis and release of dopamine and norepinephrine with respect to increased expression or activity of PSEQ. be able to. An antibody that specifically binds to the polypeptide can be used directly as an antagonist or indirectly as a targeting or delivery mechanism for delivering a drug to cells or tissues that express the polypeptide.

[0065] Antibodies to a polypeptide encoded by a PSEQ or NSEQ can also be produced using methods well known to those skilled in the art. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab
And fragments produced by Fab expression libraries. Neutralizing antibodies (ie, those that inhibit dimer formation) are particularly suitable for therapeutic use. Monoclonal antibodies to PSEQ can be made using any technique that produces antibody molecules by persistent cell lines in culture. Such techniques include, but are not limited to, hybridoma technology, human B cell hybridoma technology, and E
BV-hybridoma technology and the like are included. In addition, techniques developed for the production of chimeric antibodies can be used (see, eg, Myers, supra ). Or,
Techniques developed for the production of single-chain antibodies can be used. PSEQ or NSEQ
Antibody fragments which contain specific binding sites for the polypeptide sequence encoded by are also generated.

Various immunoassays can be used for screening to identify antibodies with the desired specificity. Many protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificity are well known to those of skill in the art.

Further, an agonist of the polypeptide encoded by PSEQ or NSEQ can be administered to a patient for the treatment or prevention of a cancer associated with reduced expression or activity of the polypeptide.

Another embodiment of the present invention relates to administering a pharmaceutical or sterile composition with a pharmaceutically acceptable carrier for all of the aforementioned therapeutic effects. Such pharmaceutical compositions may consist of the polypeptide encoded by the PSEQ or NSEQ, antibodies to the polypeptide, and mimetics, agonists, antagonists, or inhibitors. The pharmaceutical composition may be administered alone or
Alternatively, it can be administered in combination with at least one or more other drugs, such as a stabilizer, for example, administered in a sterile, biocompatible pharmaceutical carrier. Such pharmaceutical carriers include, but are not limited to, saline, buffered saline, dextrose, and water. The composition may be administered to the patient alone or in combination with other drugs, drugs or hormones.

The pharmaceutical compositions used in the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal It can be administered by administration, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal.

These pharmaceutical compositions include, in addition to the active ingredient, suitable pharmaceutically acceptable excipients and additives such as excipients and additives that facilitate the processing of the active substance into pharmaceutically usable formulations. A carrier can be included. For more information on techniques for formulation and administration may refer to the latest version of Remineto n's Pharmaceutical Sciences (Maack Publishing Co. Easson, PA).

For any formulation, a therapeutically effective amount can be administered, eg, a neoplastic cell, or a mouse,
It can be estimated initially in cell culture assays in any of the animal models such as rats, rabbits, dogs, or pigs. In addition, an appropriate concentration range and administration route can be determined using an animal model. Such information can then be used to determine the effective dose and route of administration for humans.

A therapeutically effective amount refers to an active ingredient that ameliorates a symptom or condition (eg, PSEQ or NSEQ
Or fragments thereof, antibodies of the polypeptide, as well as agonists, antagonists or inhibitors of the polypeptide). Therapeutic efficacy and toxicity can be determined by standard constraint methods in cell culture or by using experimental animals, eg, ED ₅₀ (therapeutically effective dose in 50% of the population) or LD ₅₀ (50% of the population). % Lethal dose) can be determined.

Any of the above methods of treatment may be performed, for example, in dogs, cats, cows, horses, rabbits, monkeys,
And most preferably to any subject in need of such treatment, including mammals such as humans.

[0074]

【Example】

It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described herein, which may vary. Also, the terms used in the present specification are merely intended to describe specific embodiments and are intended to limit the scope of the present invention, which is limited only by the claims. Please understand that it is not done. The following examples are illustrative of the present invention and are not intended to limit the present invention.

1. As an example of preparing a PGANNOT01 cDNA library, preparation of a PGANNOT01 library will be described. PGANNOT01 cDNA
The library was created using poly-A RNA isolated from paraganglial tumor tissue taken from the intraperitoneal region of a 46-year-old Caucasian male during exploratory laparotomy. Pathologically, it showed benign paraganglioma and was associated with clear cell grade 2 renal cell carcinoma that did not invade the capsule. Family history includes cerebrovascular disease, atherosclerotic coronary artery disease, myocardial infarction, and type II diabetes.

[0076] Frozen tissue was subjected to a Polytron homogenizer (PT-3000; Brinkmann Instruments, Westbud).
ry, NY) and homogenized and dissolved in guanidinium isothiolianate solution. The lysate was analyzed by BL8-70M ultracentrifuge (Beckmann Instruments).
Centrifugation was performed using a W28 rotor on a 5.7 M CsCl cushion at 25,000 rpm for 18 hours at ambient temperature. RNA was extracted twice with acidic phenol (pH 4.0) and precipitated with 0.3 M sodium acetate and 2.5 volumes of ethanol using an RNA isolation protocol (Stratagene, La Jolla CA), followed by DEPC-treated water. And treated with DNase at 37 ° C. for 15 minutes. The reaction was stopped with an equal volume of acidic phenol, and mRNA was isolated using OLIGOTEX kit (QIAGEN, Chatsworth CA) and used for cDNA library construction.

RNA was prepared using the SUPERCRIPT Plasmid System (Life Technologies, Gaithersburg MD)
The cDNA was ligated to the pSport I (Life Technologies) plasmid, following the recommended protocol described above. Next, the plasmid was transferred to DH5α competent cells (Life Techno
logies).

2 Isolation and Sequencing of cDNA Clones Plasmid DNA was released from the cells and it was purified using the MINIPREP kit (Edge Biosyste
ms, Gaithersburg MD). This kit consists of a 96-well block with reagents for 960 purifications. We used the recommended protocol but changed the following: (1) Each of the 96 wells was filled with only 1 ml of sterile Terrific Broth (Life Technologies) containing 25 mg / L carbenicillin and 0.4% glycerol.
(2) After inoculation, the bacteria were cultured for 24 hours and then lysed with 60 μl of lysis buffer. (3) Before adding the contents of the block to the primary filter plate, use GS-6R (B
The centrifugation process using an eckman Coulter) was performed at 2900 rpm for 5 minutes. (
4) The optional step of adding isopropanol to TRIS buffer was not performed routinely. After the final step of the protocol, samples were transferred to 96-well blocks for storage.

Other methods for purifying plasmid DNA include the MAGIC MINIPREPS system (P
romega, Madison WI) or QIAWELL-8 Plasmid, QIA WELL PLUS DNA and QIA WELL
Includes the use of ULTRA DNA purification systems (Qiagen).

The cDNA was obtained from four DNA ENGINE thermal cyclers (PTC200; MJ Research, Watertow
n MA) in combination with the MICROLAB 2200 system (Hamilton, Reno NV) and prepared using ABI PRISM 377 or 373 DNA Sequencing Systems (PE Biosystems).
Sequenced by the method of Sanger F and AR Coulson (1975; J Mol Biol 94: 441f).

3 Sequence Selection, Assembly, and Characterization Sequences used for co-expression analysis include EST sequences, 5 ′ and 3 ′ long read sequences (lo
ngread sequences), and full-length coding sequences. The selected assembly sequences were expressed in at least three cDNA libraries.

The assembly process is described below. The chromatogram of the EST sequence was processed and examined. Quality scores were obtained using PHRED (Ewing, B.
(1998) Genome Res. 8: 175-185; Ewing, B. and P. Green (1998) Genome Res.
8: 186-194). The edited sequence was loaded into a relational database management system (RDBMS). The first set of bins using BLAST with a product score of 50
EST sequences were clustered. All clusters of two or more sequences were generated as bins. The overlapping sequence represented in the bin corresponds to the sequence of the transcribed gene.

The assembly of the component sequences in each bin was performed using a modification of Phrap, a commonly available program for the assembly of DNA fragments (Green, P.,
University of Washington, Seattle, WA). Bins that showed 82% identity from local pairwise alignments between any consensus sequences were merged.

The bins were annotated by screening consensus sequences in each bin against public databases such as NCBI's GBPRI and GENPEPT. The annotation process involved FASTn screening against the GBPRI database at GENBANK. Hits with percent identity of 70% or more and alignment lengths of 100 base pairs or more were recorded as homologous hits. The remaining unannotated sequences were screened with FASTx against GENPEPT. E value is 1
Hits that are ^0-8 or less are recorded as homologous hits.

Next, Cross-Match and BLASTn (Green, P. University of Washington, S.L., which are programs for comparing protein and nucleic acid sequences at high speed and searching the database)
The sequence was re-clustered using eattle WA) sequentially. Any BLAST alignments between sequences with a score greater than 150 and the consensus sequence were re-aligned using cross-match. The sequence was added to the bin with the consensus sequence giving the highest Smith-Waterman score in the local alignment with at least 82% identity. Mismatched sequences generated new bins. The assembly and consensus sequence generation process was performed on the new bin.

[0086] 4 novel neurotransmitter processing related genes using any known gene 10 kinds of neurotransmitters processing specific gene used in the identification of new insights used in co-expression analysis described above Company genes were identified. These genes include L-tyrosine hydroxylase (TH), aromatic amino acid decarboxylase (AA
DC), dopamine β-hydroxylase (DBH), nicotinic acetylcholine receptor α3 subunit precursor (nAchR-α3), secretogranins I and II, Rab3a,
Includes human cocaine and amphetamine-regulated transcript (hCART), vesicular monoamine transporter 1 (hVMAT1), ARIX homeodomain protein, and the like. Other known genes that may be associated with the dopamine and norepinephrine pathway include:
Includes endothelin-converting enzyme (ECE), rap2 interacting protein 8 (RPIP8), neuron-specific growth-associated protein (SCG10), and a "δ-like" putative homologous heterologous protein (dlk).

L-tyrosine hydroxylase (TH) is the first and rate-limiting step in the synthesis of the catecholamines dopamine, norepinephrine, and epinephrine.
Catalyzes the conversion of -tyrosine to L-dopa (Nagarsu, T. (1995) Essays Bioch
em. 30: 15-35). TH activity is regulated by a variety of methods, including transcriptional, by alternative mRNA splicing, and by regulation of mRNA stability. Aromatic amino acid decarboxylase (AADC) is L-tyrosine, L-
Catalyzes the decarboxylation of tryptophan, L-phenylalanine and L-dopa. Decarboxylation of L-dopa produces dopamine (Webster and Jordan, supra ) and increases the level of AADC-enhanced dopamine synthesis in vivo .
Dopamine β-hydroxylase (DBH) converts dopamine to norepinephrine (Wu, HJ et al. (1990) J. Neurochem. 55: 97-105). The nicotinic acetylcholine receptor α3 subunit precursor (nAchR-α3) is a subunit in the ligand-gated sodium ion channel triggered by nicotine and acetylcholine. Prolonged stimulation of nAchR with nicotine or acetylcholine induces TH and DBH (Patrick, RL and JD Barchas (1976) J. Pharm
acol. Exp. Ther. 197: 181-196). Secretogranin is a vesicle-associated protein. Secretogranin I is found in norepinephrine-containing vesicles (Bre
ssler, JP et al. (1996) J. Neurosci. Res. 46: 678-685). Secretogranin II is a precursor of secretoneurin that stimulates dopamine release (Agneter, E. et al.
1995) J. Neurochein. 65: 622-625; Fiseher-Colbrie, R. et al. (1995) Prog. Neurob.
iol. 46: 49-70). Secretogranin II is produced in dopamine and norepinephrine-containing vesicles in neurons (Goodall, AR. Et al. (1997) J. Neuroc
hem. 68: 1542-1552). Rab3a is a member of the Ras GTPase family. R
ab3a binds to cored vesicles and partially coexists with DBH (Johannes, L. et al. (1994) Embo
J. 13: 2029-2037; Darchen, F. et al. (1995) J. Cell Sci. 108: 1639-1649) and one of the factors regulating Ca ²⁺ -dependent exocytosis (Johannes et al. , Supra ). Human cocaine and amphetamine-regulated transcript (hCART) is a signal peptide protein that is specifically induced in neurons exposed to cocaine and amphetamine (Douglass, J. and S. Daoud (1996) Gene 169: 241-245). Cocaine blocks reuptake of dopamine from synapses (Reith, ME et al. (19
97) Eur. J. Pharmacol. 324: 1-10) and also block the reuptake of amphetamine which induces dopamine release (Jones, SR et al. (1998) J. Neurosci. 18: 1979).
-1986). Vesicle monoamine transporter 1 (hVMAT1) packages the neurotransmitter monoamine into cored vesicles for exocytosis at the synapse (Liu, Y. et al. (1994) J.
Cell Biol. 127: 1419-1433). ARIX is a human homeodomain protein that is specifically expressed in noradrenergic DBH-positive tissues and cell lines derived from those tissues (Zeilmer, B. et al. (1995) J. Neurosci. 15: 8109-8120). . ARI
X regulates the transcriptional activity of the tyrosine hydroxylase gene promoter and the DBH promoter, and the two promoters regulate the specific expression of the catecholamine biosynthesis gene.

Endothelins ET-1, ET-2, and ET-3 are a class of secreted peptides that are processed from inactive precursor peptides. The third and final processing steps that trigger active ET are catalyzed by members of the endothelin converting enzyme (ECE). ET is known to be a known vasoactive drug, a potential neurotransmitter, and a potential growth factor (Bames, K. and AJ Turner (1997) Neurochem. Res. 22
: 1033-1040). ET-1 has been shown to enhance the norepinephrine pressor response (Kita, S. et al. (1998) J. Cardiovasc. Pharmacol. 31 S119-S122),
ET-1 and ET-3 have also been shown to induce dopamine release (Hone
, K. et al. (1995) Life Sci. 57: 735-741; Shibaguchi, H. et al. (1997) Cell Mol. Neur.
obiol. 17: 471-481). However, the involvement of ET-2 in norepinephrine or dopamine processing has not been clarified in the literatures so far. Rap2 interacting protein 8 (RPIP8) was discovered using yeast two-hybrid screening of mouse brain cDNA using the low molecular weight GTP-binding protein Rap2 as a feed (Janoueix-Lerosey, I . Et al. (1998) Eur, J. Biochem. 252 (2): 290-298.
). Rap2 is a member of the Ras family, and another member (Rab3a) is known to be involved in exocytosis of neurotransmitters. SCG10 is a neuron-specific growth-related protein belonging to the stathmin family. SCG10 acts in spreading microtubule collapse in nerve growth cones (Riederer, BM et al. (1997) Pr.
oc. Nail. Acad. Sci. 91: 741-745). dlk is a “δ-like” putative homologous heterogeneous protein thought to be involved in neuroendocrine differentiation (Laborda, J. et al. (1993
) J. Biol. Chem. 268: 3817-3820). It belongs to the epidermal growth factor receptor family (Lee, YL et al. (1995) Biochim. Biophys. Acta 1261: 223-232) and is induced by growth hormone (Carlsson, C. et al. (1997) Endocrinology 138: 3940-). Three
948), regulated by ErbB3 (Edman, CF et al. (1997) Biochem. J.323: 113-1).
18).

Table 4 shows the five genes most closely related to TH, a gene involved in dopamine and norepinephrine synthesis and release. These genes are shown with their P values. The items in the columns of the table have the following meanings. (P-value): Probability that the number of coincidences observed using Fisher's exact test is coincidental. (Associated gene): Gene that shows significant co-expression with the target ( Number of occurrences; Occurs): Number of libraries in which related genes occur (Both occurrence): Number of libraries in which both target genes and co-expressed genes occur (Number of targets only; Target only) : Number of libraries that generate only target genes (Number of related genes only; Associated only): Number of libraries that generate only related genes (Non-generating number: Neither): Live where neither target genes nor related genes occur Number of rallies

[0090]

[Table 3]

As targets, TH occurs in 15 of the 522 cDNA libraries examined and has neurotransmitter processing and other known genes (eg, DBH, nAchR
, Secretogranin I, human dlk mRNA for putative homologous heterologous protein
, And a homologue of endothelin converting enzyme (ECE). The results are shown in Table 4, with an associated probability of 1.58
The range is from E- ⁸ to 1.53E- ¹³ .

The other nine known genes (AADC, DEH, nAchR-α3, secretogranins I and II
, Rab3a, hCART, hVMAT1, and ARIX), the same results were obtained. These results indicate that the co-expression analysis was successful in confirming the association between the known genes, as well as the association with the previously identified known genes, and therefore, neurotransmission such as dopamine and norepinephrine. It shows that it is effective in identifying genes related to the synthesis and release of a substance.

5 Identification of Novel Neurotransmitter Processing-Related Genes Five novel genes, known as their cDNAs and those related to dopamine and neurotransmitters in the library (Incyte Pharamaceuticals) that make up the LIFESEQ database The gene was identified by co-expression analysis. That
The five genes show a statistically significant association with known dopamine and norepinephrine-related genes as measured by Fisher's exact test.
These five novel genes are potential therapeutic proteins and targets for the treatment of diseases in which norepinephrine and dopamine deviate from their normal levels.

The novel genes were identified from a total of 41,419 assembled gene sequences from Incyte. The degree of relevance is measured by a probability value, which is 0.0000
It has a P-value cutoff of less than 1. After this process, annotations and literature searches were performed to confirm that the genes that passed the established test were strongly related to known neurotransmitter processing-specific genes. This process was repeated so that the first 41,419 genes were ultimately restricted to five neurotransmitter processing-related genes. Tables 5 to 10 show details of the identification of these five genes. These tables show the five most strongly associated genes for each target novel gene when co-expression was assessed using Fisher's exact test. The column items have the same meaning as in the fourth embodiment.

[0095]

[Table 4]

[0096] The Insight 621850 gene, which occurred in 10 of the 522 cDNA libraries examined and showed strong co-expression with known genes, was
It has been implicated in neurotransmitter processing including DBH, somatostatin I and II as shown in Table 5. Gene 621850 showed a strong association with SCG10. SCG10 was shown in Example 4 to be strongly associated with known genes involved in dopamine synthesis and release. These results are consistent with the view that gene 621850 is involved in neurotransmitter processing.

[0097]

[Table 5]

[0098] Incyte gene 62, which occurred in 14 of the 522 cDNA libraries examined and showed strong co-expression with several known genes
5839 is DBH, nAchR, type 4 TH, and secretogranin I and II as shown in Table 6.
And the synthesis and release of dopamine and norepinephrine. Gene 625839 also showed a strong association with human poly (ADB-ribose) polymerase PARP expressed during Drosophila development (Uchida, K. et al. (1993) 90: 3481-3485).
). These results are consistent with the view that gene 625839 is involved in neurotransmitter processing.

[0099]

[Table 6]

Incyte gene 2405 that occurred in three of the 522 cDNA libraries examined and showed strong co-expression with several known genes
140 was associated with neurotransmitter processing, including DBH, nAchR, type 4 TH, and AADC as shown in Table 7. Gene 2405140 is also the expected nematode protein F
It showed a strong association with the homolog to 25H2.8. These results indicate that gene 2405140
Is consistent with the view that is involved in neurotransmitter processing.

[0101]

[Table 7]

[0102] Insight 28 genes that occurred in 12 of the 522 cDNA libraries examined and showed strong co-expression with several known genes
23339 was involved in neurotransmitter processing, including secretogranins I and II, hVMAT1, and type 4 TH as shown in Table 8. These results indicate that gene 2823
Consistent with the view that 339 is involved in neurotransmitter processing.

[0103]

[Table 8]

The Incyte gene 2825 which occurred in three of the 522 cDNA libraries examined and showed strong co-expression with several known genes
861 was involved in neurotransmitter processing, including DBH and TH type 4 as shown in Table 9. Gene 2858661 was also shown to be related to blood coagulation factor human factor X. These results are consistent with the view that gene 2828661 is involved in neurotransmitter processing.

6. Novel Neurotransmitter Processing-Related Genes Each of the nucleic acids comprising the nucleic acid sequence of SEQ ID NOs: 1-5 of the present invention was identified from Incyte clone numbers 621850, 625839, 2405140, 2823339, and 2825861. , According to the third embodiment. The nucleic acid sequences were translated and, where possible, the reading frame determined. The full-length protein sequence SEQ ID NO: 6 was obtained after determining its coding frame from SEQ ID NO: 4. SEQ ID NO: 1-6 is BLAST
And annotated according to Example 7 using other motif search tools against a database of known molecules.

SEQ ID NO: 1 showed 99% sequence identity with human brain specific mRNA KIAA0604 (g3043731). Furthermore, the peptide sequence translated from SEQ ID NO: 1 showed about 77.3% sequence homology with the gene encoding endothelin converting enzyme 2, ECE-2, from Bos taurus . As shown in Example 4, ECE is known to be involved in the pathway of neurotransmitter processing.

SEQ ID NO: 2 shows 60% sequence identity with the human brain-specific gene KIAA0604 (g3403731) in nucleic acids from about position 80 to about position 1818, and from about 144 to about 1760 Human for endothelin converting enzyme 1, ECE-1 (g1197803) in nucleic acids
It showed 61% sequence identity with mRNA. ECE-1 is a metalloprotease that is known to be involved in new station transmitter processing.

[0108] SEQ ID NO: 4 is a homologue to the intermediate filament (IF) protein in nucleic acids 332 to 1393, a human heavy neurofilament.
It showed about 58% sequence identity with the gene encoding the filament) subunit, NF-H (g35028). The corresponding amino acid sequence SEQ ID NO: 6 is 210 amino acids long, has one potential N-glycosylation site at residue N16 and residues S21, S100, and S1
14 potential phosphorylation sites by casein kinase II at 14, one potential glycosaminoglycan attachment site at residue S110, and two potential phosphorylation sites by protein kinase C at residues S27 and S110. Have.

7. Homology Search for Neurotransmitter-Related Genes The polynucleotide sequence SEQ ID NO: 1-5, and the polypeptide sequence SEQ ID NO: 6 were compared against databases obtained from sources such as, for example, GenBank, SwissProt. I asked. In these databases, which contain annotated sequences already identified, the Basic Local Alignment Tool (BLAST; Altschul, SF et al. (1990) J. M.
ol. Biol. 215: 403-410) and Smith-Waterman alignment (Smith, T et al. (1992) P
rotein Engineering 5: 35-51) to search for regions of similarity. BLAST
For sequence matches, and only those that met the probability threshold of less than 10 ⁻²⁵ for nucleotide sequences and less than 10 ⁻⁸ for polypeptide sequences were reported.

[0110] The polypeptide sequences were used in MOTIFS, SPSCAN, BLIMPS, and hidden Markov models (HM
We also analyze patterns of known motifs using protocols based on M).
Was. MOTIFS (Genetics Computer Group, Madison, WI) is a Prosite Dictionary
 of Protein Sites and Patterns (Bairoch, A. et al. (1997) Nucleic Acids Res. 2
5: 217-221) to a pattern corresponding to that defined in
Search sequences and display a summary of the patterns found and their corresponding literature
. SPSCAN (Genetics Computer Group, Madison, WI) uses the weighted matrix method
(Nielson, H. et al. (1997) Prot. Eng. 10: 1-6)
Search the array Hits with a score of 5 or more are considered. BLIMPS weighted
Using a matrix analysis algorithm, the polypeptide sequence and BLOCKS (PROSITE
Database (Henikoff, S. and G. J. Henikoff (1991) Nucleic Acids Res. 1
9: 6565-6572; Bairoch et al.,Above), Compiled from 3-60 amino acids
Database consisting of segments or blocks of short amino acids)
Sequence and PRINTS (such as SwissProt, GenBank, PIR, and NRL-3D).
Fingerprint data based on non-redundant sequences obtained from source
Base: Attwood, T. K. et al. (1997) J. Chem. Inf. Comput. Sci. 37: 417-424)
Search for sequence similarity between the sequences included in. For the purposes of the present invention, BLIM
By PS search, cutoff score is 1000 or more, cutoff probability value is 1.0 × 10
^-3The above agreement is reported. HMM-based protocols are based on stochastic methods
Search for the common primary structure of a gene family in a protein sequence (
Eddy, S.R. (1996) Cur.Opin. Str. Biol. 6: 361-365; Sonnhammer, E.L.L.
(1997) Proteins 28: 405-420). Cutoff score is in the range of 10-50 bits
More than 500 known protein families have been selected for use in the present invention.
Was.

8. Labeling and Use of Hybridization Probes Oligonucleotides were designed using software well known to those skilled in the art, such as OLIGO 4.06 (National Bioscience), and 50 pmol of each oligomer, 250 μCi of [γ- ³² P]
Adenosine triphosphate (Amersham, Pharmacia Biothech, Piscataway NJ) and T
Label by combining 4 polynucleotide kinases (NEN Life Science Products, Boston MA). Label the labeled oligonucleotide with SEPHADEX G-25
Substantially purified using ultrafine resin columns (Amersham Pharmacia Biotech). Aliquots containing per minute 10 ⁷ counts of labeled probe, endonuclease AseI, Bgl II, Eco RI, Pst I, Xba I or Pvu II (NEN Life Science Product
s) in a hybridization analysis using a typical membrane of human genomic DNA digested with one of the above.

The DNA from each digest was fractionated on a 0.7% agarose gel and treated with a nylon membrane (NYTRAN
PLUS, Sebleicher & Schuell, Durham NH). Hybridization 4
Perform at 0 ° C. for 16 hours. The blot is washed sequentially at room temperature under increasingly stringent conditions up to 0.1 x aqueous sodium citrate and 0.5% sodium dodecyl sulfate to remove non-specific signals. XOMAT AR film (Eastman Kodak, R
ochester NY) is exposed to the blot for several hours before comparing the hybridization patterns.

9. Acidic polyacrylamide gel electrophoresis (PAGE) of specific antibodies (see, eg, Harrington, MG (19
90) Methods Enzymol. 182: 488-495), or using substantially purified SEQ ID NO: 6 using other purification techniques to immunize rabbits according to standard protocols to generate antibodies. I do.

Alternatively, the amino acid sequence is analyzed using LASERGENE software (DNASTAR Madison WI) to determine a region having high immunogenicity, and a corresponding oligopolypeptide is synthesized, which is then used by those skilled in the art. The antibody is produced by the method described above. Details of how to select a suitable epitope, such as an epitope near the C-terminus or in a hydrophilic region, are described in the literature of those skilled in the art. Usually, an ABI 431A Peptide Synthesizer using Fmoc chemistry (Perkin-Elmer)
To enhance immunogenicity by binding to KLH (Sigma, St. Louis, MO) by reaction using MBS (N-maleimidobenzoyl-N-hydroxysuccinimide ester). Rabbits are immunized with the oligopeptide-KLH complex in Freund's complete adjuvant. The resulting antiserum is, for example, by binding the peptide to plastic, blocking with 1% BSA, reacting with rabbit antiserum, washing, and further reacting with radioactive iodine-labeled goat anti-rabbit IgG to produce the anti-peptide. Tested for activity.

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) A61P 25/08 A61P 25/18 4C084 25/16 C07K 14/47 4C086 25/18 14/705 4H045 C07K 14 / 47 16/18 14/705 16/28 16/18 16/40 16/28 C12N 1/15 16/40 1/19 C12N 1/15 1/21 1/19 9/02 1/21 9/88 5 / 10 C12Q 1/68 A 9/02 G01N 33/53 M 9/88 33/566 C12Q 1/68 C12P 21/08 G01N 33/53 C12N 15/00 ZNAA 33/566 5/00 A // C12P 21/08 A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, M , MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD) , RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG , MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Volkmouth, Wayne 940, California, United States 25. Menlo Park # 1 Roble Avenue 783 (72) Inventor Klingler, Todd M. 94070 San Carlos Dovercourt, California, USA 28 F Term (reference) 4B024 AA01 AA11 BA07 BA08 BA63 BA80 CA04 CA09 GA11 HA12 4B050 CC03 DD11 LL01 4B063 QA01 QA19 QQ02 QQ03 QQ43 QQ44 QR32. ZC022 ZC032 ZC412 4C086 AA01 EA16 MA01 MA02 MA03 MA04 MA05 NA14 ZA02 ZA06 ZA12 ZA15 ZA18 ZA20 ZA81 ZB02 ZB05 ZB26 ZC02 ZC03 ZC41 4H045 AA10 AA11 AA30 BA10 CA40 DA50 DA21 FA72 DA89

Claims (11)

[Claims] 1. A substantially generated polynucleotide comprising a gene that is co-expressed with one or more neurotransmitter processing-specific genes in a plurality of biological samples, said polynucleotide comprising a neurotransmitter processing-specific gene. Each of the target genes is L-tyrosine hydroxylase (TH), aromatic amino acid decarboxylase (AADC), dopamine β-hydroxylase (DBH), nicotinic acetylcholine receptor α3 subunit precursor (nAchR-α3), secretogla Nin I and II, R
A polynucleotide selected from the group consisting of ab3a, human cocaine and amphetamine-regulated transcript (hCART), vesicular monoamine transporter 1 (hVMAT1), and ARIX homeodomain protein. 2. The polynucleotide according to claim 1, wherein the polynucleotide comprises: (a) a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 5; and (b) a polynucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 6. (C) a polynucleotide sequence complementary to the polynucleotide sequence of (a) or (b), and (d) hybridizing with the polynucleotide of (a), (b) or (c). The polynucleotide of claim 1, comprising a polynucleotide sequence selected from a probe and a probe. 3. A substantially generated polynucleotide comprising a gene product of a gene that is co-expressed with one or more neurotransmitter processing-specific genes in a plurality of biological samples, wherein said polynucleotide is a neurotransmitter. Each of the substance processing specific genes includes L-tyrosine hydroxylase (TH), aromatic amino acid decarboxylase (AADC), dopamine β hydroxylase (DBH), nicotinic acetylcholine receptor α3 subunit precursor (nAchR-α3) Secretogranin I and II, Rab3a, human cocaine and amphetamine-regulated transcript (hCART),
A substantially generated polynucleotide selected from the group consisting of vesicular monoamine transporter 1 (hVMAT1), and ARIX homeodomain proteins. 4. The polypeptide is selected from: (a) a polypeptide sequence of SEQ ID NO: 6; and (b) a polypeptide sequence comprising at least six consecutive amino acids of the polypeptide of (a). 4. The polypeptide of claim 3, comprising a polypeptide sequence. 5. An expression vector comprising the polynucleotide of claim 2. 6. A host cell comprising the expression vector according to claim 5. 7. A pharmaceutical composition comprising the polynucleotide of claim 2 together with a suitable pharmaceutical carrier. 8. A pharmaceutical composition comprising the polypeptide of claim 4 together with a suitable pharmaceutical carrier. 9. An antibody that specifically binds to the polypeptide of claim 4. 10. A method for diagnosing a disease or condition associated with altered expression of a gene that is co-expressed with one or more neurotransmitter processing-specific genes, wherein each of said neurotransmitter processing-specific genes is Are L-tyrosine hydroxylase (TH), aromatic amino acid decarboxylase (AADC), dopamine β-hydroxylase (DBH), nicotinic acetylcholine receptor α3 subunit precursor (nAchR-α3), secretogranin I and II , Rab3a, human cocaine and amphetamine-regulated transcript (hCART), vesicular monoamine transporter 1 (hVMAT1),
Selected from the group consisting of ARIX homeodomain proteins, and further comprising: (a) providing a sample containing the one or more co-expressed genes; and (b) one or more hybridization complexes. 3. a step of hybridizing the polynucleotide of claim 2 with the co-expressed gene under conditions effective to form, and (c) a step of detecting the hybridization complex. A detection process wherein the presence of the complex is correlated with the presence of the disease or condition. 11. A method for treating or preventing a disease associated with altered expression of a gene that is co-expressed with one or more neurotransmitter processing specific genes in a patient, said method comprising: Each of the genes is L-
Tyrosine hydroxylase (TH), aromatic amino acid decarboxylase (AADC)
, Dopamine β-hydroxylase (DBH), nicotinic acetylcholine receptor α
3 subunit precursor (nAchR-α3), secretogranin I and II, Rab3a, human cocaine and amphetamine regulated transcript (hCART), vesicular monoamine transporter 1 (
hVMAT1), and the ARIX homeodomain protein, comprising administering an effective amount of the pharmaceutical composition of claim 7 to the patient in need of treatment to treat or prevent the disease. A method of treatment or prevention.