JP2003512840A - Rat brain-derived nucleic acid molecules and programmed cell death models - Google Patents

Abstract

(57) Abstract The present invention relates to a human homolog of a nucleic acid molecule derived from rat brain and a programmed cell death expression library. These molecules can constitute a microarray of expressed sequences useful for analyzing gene expression in various biological contexts, including development, differentiation, and disease, both in vivo and in vitro. Nucleic acid molecules are useful for diagnosis, treatment, and drug discovery. Nucleic acid molecules are useful for creating microarrays for transcription profiling. The invention further provides peptides encoded by the nucleic acid molecules useful in methods of diagnosis, treatment, and drug discovery. The invention particularly relates to nucleic acid molecules involved in programmed cell death.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to rat brain-derived nucleic acid molecules and programmed cell death expression libraries. Also, vectors for making and using the novel molecules of the invention,
Host cells and methods are also provided.

BACKGROUND OF THE INVENTION Much effort has been expended by modern scientific research organizations to identify and sequence genes, particularly human genes. The identification of genes and knowledge of their nucleic acid sequences enables many scientific and commercial advances in both research and diagnostic and therapeutic applications. For example, advances in gene identification and sequencing have enabled the production of products encoded by these genes, eg, by recombinant and synthetic means. In addition, the identification of genes and the products they encode may provide important information about the mechanism of the disease and provide new diagnostic tests and therapeutic treatments for the diagnosis and treatment of disease. Thus, gene identification and sequencing provides valuable information and compositions for use in biotechnology and pharmaceutical engineering.

In multicellular organisms, homeostasis is maintained by balancing the rate of cell proliferation with the rate of cell death. Cell proliferation is affected by the expression of many growth factors and proto-oncogenes, which typically promote progression through the cell cycle. In contrast, many events, including expression of tumor suppressor genes, can lead to arrest of cell proliferation.

In differentiated cells, a particular type of cell death called apoptosis occurs when the internal suicide program is activated. The program can be initiated by a variety of external signals, as well as signals generated within the host cell in response to, for example, genetic damage. Dying cells are eliminated by phagocytes without an inflammatory response.

Programmed cell death (PCD) is a highly regulated process (Wils
on (1998) Biochem. Cell. Biol. 76: 573-582
). This death signal is then transformed via various signaling pathways that assemble into a caspase-mediated degradation cascade, which activates late effector activities of morphological and physiological aspects of apoptosis, including DNA fragmentation and cytoplasmic aggregation. Will occur. Furthermore, regulation of programmed cell death is regulated by energy, redox and ionic homeostasis in mitochondria (Kroemer (199
8) Cell Death and Differentiation 5: 5
47), and / or regulation of the cell cycle in the nucleus and cytoplasm (Choisy-Rossi and Yonish-Rouach (1
998) Cell Death and Differentiation 5
: 129-131; Dang (1999) Molecular and Cel.
tubular Biology 19: 1-11; and Kasten and G.
iordano (1998) Cell Death and Differen
5: 132-140). Many mammalian genes that regulate apoptosis are originally Caenorhabdit
is elegans or Drosophila melanogaster
Were identified as homologues of genes genetically identified in, or as human oncogenes. Other programmed cell death genes have been found by domains homologous to known motifs that mediate protein-protein interactions within the programmed cell death pathway (eg, death domains).

[0006] The mechanisms that mediate apoptosis include activation of endogenous proteases, loss of mitochondrial function, and structural changes (eg, cytoskeleton disruption, cell contraction, membrane vesicle formation, and DNA degradation). Due to condensation of nuclei). The various signals that cause apoptosis can trigger these events by focusing on the normal cell death pathway, which is regulated by the expression of highly conserved genes.

Caspases, a cysteine protease with specificity for the substrate cleavage site aspartate, are central to the apoptotic program. These proteases are responsible for the degradation of cellular proteins, which leads to the morphological changes seen in apoptotic cells. One of the human caspases was previously known as interleukin-1β (IL-1β) converting enzyme (ICE), and cysteine protease is responsible for the processing of pro-IL-1β into active cytokines. Overexpression of ICE in rat-1 fibroblasts induces apoptosis (Miura et al. (1993) Cell 75: 653).

Many caspases and proteins that interact with caspases possess a domain of 60 amino acids called the caspase recruitment domain (CARD). Apoptotic proteins can bind to each other via their CARDs. Different subtypes of CARD confer binding specificity,
It can regulate the activity of various caspases (Hofmann et al. (1997) TIBS 22: 155).

The functional significance of CARD has recently been demonstrated in two publications. Duan
Et al. (1997) Nature 385: 86 CAR at the N-terminus of RAIDD.
Deletion of D, a newly identified protein associated with apoptosis, showed the abrogated ability of RAIDD to bind caspases. In addition, Li et al. (1997)
Cell 91: 479 is a protease activator of apoptosis-1 (Ap
It has been shown that the N-terminal 97 amino acids of af-1) are sufficient to confer caspase-9-binding capacity.

Thus programmed cell death (apoptosis) is a normal physiological activity required for proper differentiation in all vertebrates. Defects in the apoptotic program result in disorders including, but not limited to, neurodegenerative disorders, cancer, immune deficiency, heart disease and autoimmune diseases (Thompson et al. (1995) Sci.
ence 267: 1456).

In vertebrate species, the mechanism of programmed cell death of neurons has various developmental roles (establishment of appropriate synaptic connections (Openpenheim et al. (1991) Annu).
al Rev. Neuroscience 14: 453-501), quantitative agreement of pre- and post-synaptic population sizes (Herrup et al. (1987) J. Neur.
osci. 7: 829-836), and the formation of neuronal circuits both during development and in adults (Bottjer et al. (1992) J. Neuro.
biol. 23: 1172-1191), including removal of neuronal precursors that impair).

Inappropriate apoptosis is associated with various neurodegenerative diseases, such as Alzheimer's disease (
Loo et al. (1993) Proc. Natl. Acad. Sci. 90: 7951
-7955), Huntington's disease (Porterra-Cailliau et al. (1
995) J. Neurosc. 15: 3775-3787), amyotrophic lateral sclerosis (Rabizadeh et al. (1995) Proc. Natl. Acad. Sci.
． 92: 3024-3028), and pectoralis brevis atrophy (Roy et al. (1995) C.
ell 80: 167-178)).

Furthermore, inappropriate expression of genes associated with apoptosis is associated with carcinogenesis.
Thus, some "oncogenes" have been shown to be actually associated with apoptosis (eg, in the Bcl family).

Therefore, genes associated with apoptosis are important targets for therapeutic treatment. Therefore, it is important to identify new genes associated with apoptosis or to discover if known genes function in this process.

Nucleic acid probes have long been used to detect complementary nucleic acid sequences in a nucleic acid of interest (“target” nucleic acid). In some assay formats, the nucleic acid is tethered (ie, covalently) to a solid support. Arrays of nucleic acid sequences immobilized on a solid support have been used to detect specific nucleic acid sequences in target nucleic acids. See, for example, PCT Patent Publication Nos. WO 89/10977 and 89/11548. Others have proposed the use of multiple nucleic acid sequences to provide the complete nucleic acid sequence of a target nucleic acid, using a method that uses an array of immobilized nucleic acid sequences for this purpose. U.S. Pat. Nos. 5,202,231 and 5,002,867, and PCT Patent Publication No. WO 93/1712.
See 6.

Advances in certain microarray technologies have provided methods for making very large arrays of nucleic acid sequences in very small physical arrays. US Patent No. 5,
143,854, and PCT Patent Publication Nos. WO 90/15070 and 9
2/10092, each of which is incorporated herein by reference. U.S. patent application Ser. No. 082,937, filed June 25, 1993, discloses a sequence of sequences that can be used to provide the complete sequence of a target nucleic acid and to detect the presence of a nucleic acid containing a particular nucleotide sequence. A method of making an array is described. Therefore, microfabricated arrays of multiple nucleic acid sequences, called "DNA chips," offer great promise for a wide variety of applications.

SUMMARY OF THE INVENTION The present invention is based on the identification of a novel nucleic acid molecule from rat brain and a programmed cell death cDNA library.

Accordingly, in one aspect, the invention provides a group consisting of the sequences set forth in SEQ ID NOs: 1-6, 8 and 10 and the complement of the sequences set forth in SEQ ID NOs: 1-6, 8 and 10. There is provided an isolated nucleic acid molecule comprising a nucleotide sequence selected from.

The present invention also provides the isolation of any of the sequences set forth in SEQ ID NOs: 1-6, 8 and 10 and the complement of the sequences set forth in SEQ ID NOs: 1-6, 8 and 10. A fragment or portion that has been In some embodiments, the fragment is useful as a probe or primer, and / or at least 15, at least 18, or at least 20, 22, 25, 30.
, 35, 50, 100, 200 or more nucleotides in length.

In another embodiment, the invention provides the group consisting of the sequences set forth in SEQ ID NOs: 1-6, 8 and 10 and the complement of the sequences set forth in SEQ ID NOs: 1-6, 8 and 10. At least about 60% identical to a nucleotide sequence selected from
Nucleotide sequences that are about 65% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, or about 99% or more identical. An isolated nucleic acid molecule comprising:

In another embodiment, the invention comprises the sequence of SEQ ID NOS: 1-6, 8 and 10 and the complement of the sequences set forth in SEQ ID NOs: 1-6, 8 and 10. Provided are isolated nucleic acid molecules that hybridize to selected nucleotide sequences under conditions of high stringency.

The present invention further provides a nucleic acid vector containing the above-described nucleic acid molecule. In one embodiment, the nucleic acid molecule of the invention is operably linked to at least one expression control element.

The present invention further includes host cells (eg, bacterial cells, fungal cells, plant cells, insect cells and mammalian cells) containing the nucleic acid vectors described above.

In another aspect, the invention provides isolated gene products, proteins and polypeptides encoded by the nucleic acid molecules of the invention.

The invention further provides antibodies (monoclonal antibodies, or antigen-binding fragments thereof) that selectively bind to the isolated proteins and polypeptides of the invention.

The invention also provides for the preparation of proteins and polypeptides encoded by the isolated nucleic acid molecules described herein by culturing host cells containing the vector molecules of the invention. Provide a way.

Further, the present invention provides for the contacting of a sample with an agent (eg, an antibody or nucleic acid molecule) suitable for the specific detection of a nucleic acid sequence, protein or polypeptide in a biological sample (eg, Methods are provided for assaying for the presence of a nucleic acid sequence, protein or polypeptide of the invention (in a tissue sample).

The invention also provides a kit comprising a nucleic acid probe which hybridizes to the nucleotide sequence of claim 1 and instructions for use, and a polypeptide of claim 10. Kits with binding agents and instructions for use are provided.

DETAILED DESCRIPTION OF THE INVENTION I. Isolated Nucleic Acid Molecules The present invention provides an in vitro model of rat brain and programmed cell death (a neuronal apoptosis regulatory candidate.
the discovery and isolation of nucleic acid molecules expressed in aerated candies) or NARC), and their human homologs. The sequences of these human homologues are, in detail, SEQ ID NO: 1 (human NARC 9B), 2 (human NRC).
ARC 8B), 3 (human NARC 2A), 4 (human NARC 16B), 5
(Human NARC 10C), 6 (human NARC 1C), 8 (human NARC 1
A), and 10 (human NARC 25).

Suitably, the isolated nucleic acid molecule of the invention is RNA (eg mRNA).
Or it may be DNA, such as cDNA and genomic DNA. DNA molecules can be double-stranded or single-stranded; single-stranded RNA or DNA can be either the coding (ie, sense) strand or the non-coding (ie, antisense) strand. The nucleic acid molecule may comprise all or part of the coding sequence of the gene of the invention. In addition, the nucleic acid molecule can be fused to a marker sequence, such as a sequence encoding a polypeptide to aid in the isolation or purification of the polypeptide. Examples of such a sequence include a sequence encoding a glutathione-S-transferase (GST) fusion protein, and influenza-derived hemagglutinin A (hem).
Examples include, but are not limited to, sequences encoding an aglutin A) (HA) polypeptide marker.

An “isolated” nucleic acid molecule, as used herein, is a nucleic acid molecule that has been separated from the nucleic acids that normally naturally flank the nucleic acid molecule. Genome D
With respect to NA, the term "isolated" refers to a nucleic acid molecule whose genomic DNA is separated from the chromosome with which it is naturally associated. For example, an isolated nucleic acid molecule is less than about 5 kb, less than about 4 kb, less than about 3 kb, less than about 2 kb, less than about 1 kb, about 0 adjacent to the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived. It may comprise less than 0.5 kb or less than about 0.1 kb of nucleotides.

Further, an isolated nucleic acid of the invention (eg, a cDNA or RNA molecule)
Can be substantially free of other cellular material or culture medium if produced by recombinant techniques, or chemical precursors or other chemicals if chemically synthesized. It can be substantially free of material. However, the nucleic acid molecule may be fused to other coding or regulatory sequences and still be considered isolated.
In some cases, the isolated material forms part of a composition (eg, crude extract containing other materials), buffer system or reagent mixture. Under other circumstances, this material may be purified to essentially homogeneity as determined, for example, by PAGE or column chromatography (eg, HPLC). Preferably, the isolated nucleic acid comprises the presence of at least about 50, 80, or 90% (on a molar basis) of all macromolecular species.

Furthermore, recombinant DNA contained in the vector is included in the definition of "isolated" as used herein. Also, isolated nucleic acid molecules include recombinant DNA molecules in heterologous host cells, and partially or substantially purified DNA molecules in solution. "Isolated" nucleic acid molecules also include in vivo and in vitro RNA transcripts of the DNA molecules of the present invention.

The invention further provides variants of the isolated nucleic acid molecules of the invention. Such variants may occur naturally (eg, allelic variants (same locus),
It can be constructed by homologs (different loci), and orthologs (different organisms), or by recombinant DNA methods or chemical synthesis. Such non-naturally occurring variants can be made using well-known mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. Thus, variants can include nucleotide substitutions, deletions, inversions, and / or insertions in either or both the coding and non-coding regions of the nucleic acid molecule.
Furthermore, the variations can generate both conservative and non-conservative amino acid substitutions.

Typically, the variants have substantial identity with a nucleic acid molecule selected from the group consisting of the sequences set forth in SEQ ID NOs: 1-6, 8 and 10 and their complements. Particularly preferred is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least 96 with the nucleic acid molecules described herein. %, At least about 97%
, Nucleic acid molecules and fragments having at least about 98%, or at least about 99% or greater identity.

Such a nucleic acid molecule hybridizes under stringent conditions to a nucleotide sequence selected from the group consisting of the sequences shown in SEQ ID NOs: 1 to 6, 8 and 10 and their complements. Since it can be soybean, it can be easily identified. 1
In one embodiment, the variant has high stringency hybridization conditions (eg, for selective hybridization) to a nucleotide sequence selected from the sequences set forth in SEQ ID NOs: 1-6, 8 and 10. ) Under, hybridize.

As used herein, the term “hybridize under stringent conditions” describes conditions for hybridization and washing. Stringent conditions are known to those of skill in the art and are known to Current Prot.
ocols in Molecular Biology, John Wile
y & Sons, N.Y. Y. (1989), 6.3.1-6.3.6. Aqueous and non-aqueous methods are described in this reference, and either can be used. An example of preferred stringent hybridization conditions is hybridization in 6 × sodium chloride / sodium citrate (SSC) at about 45 ° C., followed by 0.2 × SSC, 0.1% SDS at 50 ° C.
One or more washes in. Another example of stringent hybridization conditions is hybridization in 6 × sodium chloride / sodium citrate (SSC) at about 45 ° C., followed by 0.2 × SSC, 0.1% S at 55 ° C.
One or more washes in DS. A further example of stringent hybridization conditions is 6 × sodium chloride / sodium citrate (S
SC), then 0.2 x SSC, 0 at 60 ° C.
． One or more washes in 1% SDS. Preferably, stringent hybridization conditions are hybridization in 6 × sodium chloride / sodium citrate (SSC) at about 45 ° C., followed by 0.2 × S at 65 ° C.
One or more washes in SC, 0.1% SDS. Used when practitioners are uncertain as to particularly preferred stringency conditions (and which conditions should be applied to determine whether a molecule is within the hybridization limits of the invention). Conditions to be met are 0.5 M sodium phosphate, 7% SDS at 65 ° C., followed by one or more washes in 0.2 × SSC, 1% SDS at 65 ° C. The hybridization step may be carried out for 4, 8, 12 or 16 hours, and the wash step is generally 15 minutes to 30 minutes long.

The percent identity of two nucleotide or amino acid sequences can be determined by aligning the sequences for optimal comparison purposes (eg, a gap may be introduced in the sequence of the first sequence). . The nucleotides or amino acids at corresponding positions are then compared. And the percent identity between two sequences is a function of the number of identical positions shared by these sequences. In particular embodiments,
The length of sequences aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the reference sequence. Is the length. The actual comparison of two sequences can be accomplished by well known methods (eg, using mathematical algorithms). A preferred non-limiting example of such a mathematical algorithm is K
arlin et al. (1993) Proc. Natl. Acad Sci. USA, 9
0: 5873-5877. Such an algorithm is
schul et al. (1997) Nucleic Acids Res. , 25:38
9-3402 and the NBLAST and XBLAST programs (
It is incorporated in version 2.0). BLAST and Gapped BL
When using the AST program, the individual programs (eg NBLAS
The default parameters of T) can be used. http: // www. ncb
i. nlm. nih. gov. checking ... In one embodiment, the parameters for sequence comparisons are: score = 100, wordlength (wordl)
length) = 12, but this can be varied (eg, W = 5 or W = 20).

Another preferred non-limiting example of a mathematical algorithm used for sequence comparison is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the CGC sequence alignment software package. When using the ALIGN program to compare amino acid sequences, the PAM120 weight residue table (PAM120 weight residue) is used.
table), gap length penalty
ty) 12, and gap penalties 4 may be used. Additional algorithms for sequence analysis are known in the art, and Torellis and Robotti (1994) Comput.
． Appl. Biosci. 10: 3-5 as described in ADVANCE.
And ADAM; and Pearson and Lipman (1988) PN.
FA, described in AS, 85: 2444-8.

In another embodiment, the percent identity between two amino acid sequences is BLOSU.
Either an M 63 matrix or a PAM 250 matrix, and 12,
10, 8, 6, or 4 gap weights and 2, 3,
Or GAP in a CGC software package (available at http://www.cgc.com) with a length weight of 4
It can be accomplished using a program. In yet another embodiment, the percent identity between two nucleic acid sequences is determined by the G in the CGC software package (available at http://www.cgc.com) with a gap weight of 50 and a length weight of 3.
It can be achieved using the AP program.

The invention also provides a nucleotide sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 1-6, 8 and 10 and the complement of the sequences set forth in SEQ ID NOs: 1-6, 8 and 10. In contrast, an isolated nucleic acid comprising a fragment or portion that hybridizes under conditions of high stringency is provided. In one embodiment, the nucleic acid is a nucleotide selected from the group consisting of the sequences set forth in SEQ ID NOs: 1-6, 8 and 10 and the complement of the sequences set forth in SEQ ID NOs: 1-6, 8 and 10. Consists of fragments of a sequence. The nucleic acid fragment of the invention is at least about 15 nucleotides, preferably at least about 18, 20, 23 or 2.
It is 5 nucleotides and can be 30, 40, 50, 100, 200 or more nucleotides in length. Longer fragments (eg, 30 nucleotides or more in length) encoding the antigenic proteins or polypeptides described herein are useful. In addition, the nucleotide sequences described herein can also be contigated (eg, overlapped or linked) to produce longer sequences (eg, http://bozeman.mbt.washington.edu/p).
rap. docs / phrap. html).

In a related aspect, the nucleic acid fragments of the invention are used as probes or primers in the assays as described herein. "Probe" is
An oligonucleotide that hybridizes in a base-specific manner to a complementary strand of a nucleic acid. Such probes include Nielsen et al. (1991) Sci.
and the polypeptide nucleic acids as described in ence, 254, 1497-1500. Typically, the probe is at least about 1 of a nucleic acid selected from the group consisting of the sequences set forth in SEQ ID NOs: 1-6, 8 and 10, and their complements.
5, typically about 20-25, and more typically about 40, 50, or 75.
It includes a region of a nucleotide sequence that hybridizes to a contiguous nucleotide under highly stringent conditions. More typically, the probe further comprises a label (eg, radioisotope, fluorescent compound, enzyme, or enzyme cofactor).

As used herein, the term “primer” is template-directed using well-known methods (eg, PCR, LCR), including but not limited to the methods described herein. A single-stranded oligonucleotide that acts as a starting point for sexual DNA synthesis. The appropriate length of a primer depends on the particular application, but typically ranges from about 15-30 nucleotides. The term "primer site" refers to the region of target DNA to which a primer hybridizes. The term "primer pair"
Is a 5 '(upstream) primer that hybridizes to the 5'end of the nucleic acid sequence to be amplified, and 3' (downstream) to hybridize to the complement of the sequence to be amplified.
A set of primers including a primer.

The nucleic acid molecules of the present invention (eg, the nucleic acid molecules described above) use standard molecular biology techniques and the sequence information provided in the sequences set forth in SEQ ID NOs: 1-6, 8 and 10. Can be identified and isolated. For example, nucleic acid molecules are SEQ ID NOs: 1-6, 8
, And 10 and the synthetic oligonucleotide primers designed based on one or more sequences provided in their complements can be amplified and isolated by the polymerase chain reaction. Generally, PCR T
technology: Principles and Application
s for DNA Amplification (edited by HA Errich,
Freeman Press, NY, NY, 1992); PCR Protocol
ols: A Guide to Methods and Applicati
ons (edited by Innis et al., Academic Press, San Diego)
, CA, 1990); Mattila et al. (1991) Nucleic Acid.
s Res. 19.4967; Eckert et al. (1991) PCR Metho.
ds and Applications, 1:17; PCR (McPhers
On et al., IRL Press, Oxford); and US Pat. No. 4,683.
, 202. Nucleic acid molecules are used as templates for cDNA, mRNA
, Or can be amplified using genomic DNA, cloned into an appropriate vector, and characterized by DNA sequence analysis.

Another suitable amplification method is ligase chain reaction (LCR) (Wu and Wa
Lace (1989) Genomics, 4: 560, Landegren et al. (1988) Science, 241: 1077), transcription amplification (K).
who et al. (1989) Proc. Natl. Acad. Sci. USA, 86:
1173), and self-sustained sequence replication (G
uatelli et al. (1990) Proc. Nat. Acad. Sci. USA,
87: 1874), as well as nucleic acid-based sequence amplification (NASBA). The latter two amplification methods are based on isothermal transcription, which produces both single-stranded RNA (ssRNA) and double-stranded DNA (dsDNA) as amplification products in a ratio of about 30: 1 or 100: 1, respectively. Includes isothermal reactions.

Amplified DNA can be radiolabeled and labeled with zap express, Z
A cDNA library in IPLOX or other suitable vector, can be used as a probe to screen mRNA. Corresponding clones can be isolated by methods known in the art to identify the correct open reading frame encoding a protein of appropriate molecular weight, and the DNA is excised in vivo (i
inserts, and cloned inserts can be sequenced in either or both directions. For example, direct analysis of the nucleotide sequence of the nucleic acid molecule of the present invention can be accomplished using well known commercially available methods. For example, Sambrook et al., Molecular Cloning, AL.
Laboratory Manual (2nd edition, CSHP, New York 1
989); Zyskind et al., Recombinant DNA Labora.
See Tory Manual (Acad. Press, 1988)).
Using these or similar methods, the protein and the DNA encoding the protein can be isolated, sequenced, and further characterized.

Antisense nucleic acids of the invention can be designed using the nucleotide sequences of the sequences set forth in SEQ ID NOs: 1-6, 8 and 10 and chemically synthesized and enzymatically using procedures known in the art. Can be constructed using a biological ligation reaction. For example, an antisense nucleic acid (eg, an antisense oligonucleotide) is a naturally occurring nucleotide, or formed to increase the biological stability of the molecule, or formed between an antisense nucleic acid and a sense nucleic acid. Using various modified nucleotides designed to increase the physical stability of the duplex
It can be chemically synthesized (eg, phosphorothioate derivatives and acridine substituted nucleotides can be used). Examples of modified nucleotides that can be used to make antisense nucleic acids include: 5-Fluorouracil, 5
-Bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl)
Uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosyleosinine, inosine, N6-isopentenyladenine, 1-methylguanine, 1- Methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wibutoxin, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil. , 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (
v), 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector in which the nucleic acid is subcloned in the antisense orientation (ie, RNA transcribed from the inserted nucleic acid is directed against the target nucleic acid of interest). Antisense direction)
.

Further, the nucleic acid molecule of the present invention may be modified at the base moiety, sugar moiety or phosphate backbone to improve, for example, the stability, hybridization or solubility of the molecule. For example, the deoxyribose phosphate backbone of nucleic acids can be modified to produce peptide nucleic acids (Hyrup et al. (1996) Bioorganic & Medic.
al Chemistry 4: 5). As used herein, the term "peptide nucleic acid" or "PNA" means that the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone, and the four natural nucleobases.
ase) refers to a nucleic acid mimic (eg, a DNA mimic) in which only the P
The neutral backbone of NA has been shown to allow specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers is described by Hyrup et al. (1996); Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93: 1467
Can be performed using standard solid phase peptide synthesis protocols as described in PNAs are conjugated, for example, by attaching lipophilic or other helper groups to PNAs to enhance their stability, specificity, or cellular uptake, by forming PNA-DNA chimeras, or It can be further modified by the use of liposomes or other techniques of drug delivery known in the art. Synthesis of PNA-DNA chimeras is described in Hyrup (1996), supra, Finn et al. (1996) Nucleic Acids Res. 24.17): 3357-.
63, Mag et al. (1989) Nucleic Acids Res. 17:59
73, and Peterser et al. (1975) Bioorganic Med.
Chem. Lett. 5: 1119.

The nucleic acid molecules and fragments of the invention may also include other accessory groups such as: Peptides (eg, for targeting host cell receptors in vivo).
, Or factors that facilitate transport across cell membranes (eg, Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86:65.
53-6556; Lemaitre et al., 1987, Proc. Natl. Aca
d. Sci. 84: 648-652; PCT Publication No. WO88 / 09810), or the blood-brain barrier (eg PCT Publication No. WO89 / 10134).
checking). In addition, the oligonucleotide may be conjugated to a hybridization-triggered cleavage agent (eg, Krol et al., 1988, BioTechniques 6: 9).
58-976), or an intercalating agent (e.g., Zon, 1).
988, Pharm. Res. 5: 539-549)).

The uses of the nucleic acids of the invention are described in detail below. In general, the isolated nucleic acid sequences can be used as molecular weight markers in Southern gels and as chromosomal markers labeled to map the location of related genes. Nucleic acid sequences may also be used to compare with endogenous DNA sequences in a patient to identify genetic disorders, and to probe and detect, for example, to hybridize and discover related DNA sequences, or known sequences from a sample. Subtract (subtr
can be used as a probe to act out). The nucleic acid sequence is further
Used as a primer for gene fingerprinting, to elicit anti-protein antibodies using DNA immunization technology, and as an antigen to elicit anti-DNA antibodies or to elicit an immune response obtain. further,
The nucleotide sequences of the invention are compatible either for identifying and expressing recombinant proteins for analytical, characterization or therapeutic use, or either constitutively, during tissue differentiation or during disease states. Can be used as a marker for the tissue in which the protein is expressed.

(II. Vector and Host Cell) Another aspect of the present invention relates to a nucleic acid vector containing a nucleic acid selected from the group consisting of the sequences shown in SEQ ID NOs: 1 to 6, 8 and 10. These vectors contain the sequences of the invention inserted in the sense or antisense orientation. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors, such as non-episomal mammalian vectors, integrate into the host cell's genome when introduced into the host cell, and thereby replicate with the host's genome. Moreover, a particular vector, the expression vector, may direct the expression of the gene to which it is operatively linked. In general, expression vectors useful in recombinant DNA technology are often plasmids (vectors).
It is in the form of. However, the invention is intended to include such other forms of expression vectors, such as viral vectors that serve equivalent functions (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses).

A preferred recombinant expression vector of the invention comprises a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which host cell the recombinant expression vector is used for expression in. It is meant to include one or more regulatory sequences selected on the basis of This regulatory sequence is operably linked to the expressed nucleic acid sequence. In a recombinant expression vector, "operably linked" allows the nucleotide sequence of interest to allow expression of the nucleotide sequence (eg, an in vitro transcription / translation system, or this vector is introduced into a host cell). In a host cell) in such a manner as to be linked to regulatory sequences. The term "regulatory sequence"
It is intended to include promoters, enhancers and other expression control elements (eg, polyadenylation signals). Such regulatory sequences are, for example, Go
eddel, Gene Expression Technology: Met
hods in Enzymology 185, Academic Pres
S. San Diego, CA (1990). Regulatory sequences include sequences that direct constitutive expression of nucleotide sequences in many types of host cells, and sequences that direct expression of nucleotide sequences only in the particular host cell (eg, tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector may depend on such factors as the choice of host cell to be transformed, the level of expression of the desired protein, etc. The expression vector of the present invention can produce a protein or peptide (including a fusion protein or a fusion peptide) encoded by the nucleic acid as described herein when introduced into a host cell.

Recombinant expression vectors of the invention may be used in prokaryotic or eukaryotic cells (eg,
It can be designed for expression of a polypeptide of the invention in bacterial cells (eg E. coli), insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel (supra). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. executed in E. coli. Fusion vectors add a number of amino acids to the protein encoded therein, usually at the amino terminus of recombinant proteins. Such fusion vectors are typically
Useful for three purposes: 1) to increase the expression of the recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) by acting as a ligand in affinity purification. To assist in the purification of. Often, in a fusion expression vector, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein, allowing the recombinant protein to be separated from the fusion moiety after purification of the fusion protein. To do. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin, and enterokinase. Glutathione S is a typical fusion expression vector.
-PGEX (Pharm) that fuses transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
acia Biotech Inc; Smith and Johnson (198).
8) Gene 67: 31-40), pMAL (New England Bi).
olabs, Beverly, Mass. ) And pRIT5 (Pharmac)
ia, Piscataway, N .; J. ) Is mentioned.

Suitable inducible unfused E. An example of an E. coli expression vector is pTrc (Am
runn et al. (1988) Gene 69: 301-315) and pET 11
d (Studier et al., Gene Expression Technology)
y: Methods in Enzymology 185, Academic
Press, San Diego, California (1990) 60-
89). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. p
Target gene expression from the ET11d vector relies on transcription from the T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is lacUV 5
Supplied by resident prophage-derived host strains BL21 (DE3) or HMS174 (DE3) carrying the T7 gn1 gene under the transcriptional control of a promoter.

E. One strategy for maximizing recombinant protein expression in E. coli is to express the protein in host bacteria with impaired ability to proteolytically cleave the recombinant protein (Gottesman, Gene).
Expression Technology: Methods in En
zymology 185, Academic Press, San Diego
o, California (1990) 119-128). Another strategy is that the individual codons for each amino acid are to modify the nucleic acid sequence of the nucleic acid inserted into the expression vector so that it is preferentially utilized in E. coli (
Wada et al. (1992) Nucl. Acids Res. 20: 2111-21
18). Such alterations of the nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

In another embodiment, the expression vector is a yeast expression vector. Yeast S.
Examples of vectors for expression in S. cerevisiae include pYepSec1.
(Baldari et al., (1987) EMBO J 6: 229-234), pM.
Fa (Kurjan and Herskowitz, (1982) Cell 30.
: 933-943), pJRY88 (Schultz et al., (1987) Gene.
54: 113-123), pYES2 (Invitrogen Corp.
ation, San Diego, CA), and pPicZ (InVitro
gen Corp. , San Diego, CA).

Alternatively, the nucleic acids of the invention can be expressed in insect cells using a baculovirus expression vector. Baculovirus vectors available for expression of proteins in cultured insect cells (eg, Sf9 cells) include pAc series (S
Smith et al. (1983) Mol. Cell. Biol. 3: 2156-2165
) And pVL series (Lucklow and Summers (1989) V
irology 170: 31-39).

In yet another embodiment, the nucleic acids of the invention are expressed in mammalian cells using mammalian expression vectors. An example of a mammalian expression vector is pCDM8
(Seed (1987) Nature 329: 840) and pMT2PC (
Kaufman et al. (1987) EMBO J 6: 187-195). When used in mammalian cells, the expression vector's control functions are often
Provided by the regulatory elements of the virus. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells, see Sambrook et al. (Supra), chapters 16 and 17.

In another embodiment, the recombinant mammalian expression vector may direct the expression of the nucleic acid preferentially in a particular cell type (eg, expressing the nucleic acid using tissue-specific regulatory elements). . Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (
Liver-specific; Pinkert et al. (1987) Genes Dev 1: 268-.
277), a lymphoid-specific promoter (Calame and Eaton (198).
8) Adv Immunol 43: 235-275), especially the T cell receptor promoter (Winoto and Baltimore (1989) EMBO.
J 8: 729-733) and immunoglobulins (Banerji et al. (198).
3) Cell 33: 729-740; Queen and Baltimore (
1983) Cell 33: 741-748), neuron-specific promoters (eg, neurofilament promoter; Byrne and Rudle).
(1989) Proc. Natl. Acad. Sci. USA 86: 5473
-5477), pancreas-specific promoter (Edlund et al. (1985) Scie.
230: 912-916), and a mammary gland-specific promoter (eg,
Milk whey promoters; U.S. Patent No. 4,873,316 and European Patent Publication No. 264,166). Developmentally regulated promoters are also included (eg, the murine hox promoter (Ke
ssel and Gruss (1990) Science 249: 374-37.
9) and the α-fetoprotein promoter (Campes and Tilgh)
man (1989) Genes Dev 3: 537-546).

The invention further provides a recombinant expression vector comprising the DNA molecule of the invention cloned into the expression vector in the antisense orientation. That is, the DNA molecule expresses an RNA molecule that is antisense to the mRNA of the present invention (DNA
(By transcription of the molecule) is operably linked to at least one expression control sequence. Regulatory sequences operably linked to the cloned nucleic acid in the antisense orientation may be chosen that direct the sustained expression of the antisense RNA molecule in various cell types. For example, viral promoters and / or enhancers, or regulatory sequences may be chosen that direct constitutive, tissue-specific, or cell-type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid, or attenuated virus, where the antisense nucleic acid is produced under the control of a high efficiency regulatory region, the activity of which is introduced into the vector. Cell type. For a discussion of the regulation of gene expression using antisense genes, see Weintraub et al., Reviews-Trends in Genetics, Volume 1 (1) 19.
See 86.

Another aspect of the present invention relates to a host cell into which the recombinant expression vector of the present invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular test cell, but to the progeny or potential progeny of such a cell. Such progeny may, in fact, not be identical to the parental cell, as the particular modification may be present in the next generation, either due to mutations or environmental influences, but is still referred to herein. Included within the scope of this term as used in.

The host cell can be any prokaryotic or eukaryotic cell. For example, the nucleic acids of the invention can be expressed in bacterial cells (eg, E. coli), insect cells, yeast or mammalian cells (eg, Chinese Hamster Ovary cells (CHO) or COS cells). Other suitable host cells are known to those of skill in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" refer to various art-recognized techniques for introducing exogenous nucleic acid (eg, DNA) into a host cell. Are intended to include and include calcium phosphate or calcium chloride co-precipitation, DEAE dextran mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (Supra) and other laboratory manuals.

For stable transfection of mammalian cells, it is known that only a fraction of the cells can integrate foreign DNA into their genome, depending on the expression vector and transfection technique used. . To identify and select for these elements, a gene encoding a selectable marker (eg, resistance to antibiotics) is generally introduced into the host cell along with the gene of interest. Preferred selectable markers include markers that confer resistance to drugs such as G418, hygromycin, and methotrexate. The nucleic acid encoding the selectable marker can be introduced into the host cell on the same vector as the nucleic acid of the invention or can be introduced on a separate vector. Cells that are stably transfected with the introduced nucleic acid can be identified by drug selection (eg, cells that have incorporated the selectable marker gene will survive but other cells will die).

Host cells of the invention (eg, prokaryotic or eukaryotic host cells in culture) can be used to produce (ie, express) a polypeptide of the invention. Accordingly, the invention further provides methods for producing a polypeptide using the host cells of the invention. In one embodiment, the method comprises subjecting a host cell of the invention, into which a recombinant expression vector encoding a polypeptide of the invention has been introduced, to production of the polypeptide in a suitable medium. Culturing to. In another embodiment, the method further comprises isolating the polypeptide from the medium or host cell.

The host cells of the invention can also be used to produce nonhuman transgenic animals. For example, in one embodiment, the host cell of the invention is a fertilized oocyte or embryonic stem cell into which the nucleic acid of the invention has been introduced. Such host cells can then be used to produce non-human transgenic animals having exogenous nucleotide sequences introduced into their genome, or homologous recombinant animals with altered endogenous nucleotide sequences. . Such animals are useful for studying the function and / or activity of its nucleotide sequence and the polypeptides encoded by that sequence, and for identifying and / or evaluating modulators of its activity. As used herein, a "transgenic animal" is a non-human animal in which one or more of the cells of the animal contains a transgene,
It is preferably a mammal, more preferably a rodent such as rat or mouse. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians and the like. The transgene integrates into the genome of the cell in which the transgenic animal develops and remains in the genome of the mature animal, directing the expression of the encoded gene product in one or more cell types or tissues of that transgenic animal. , Exogenous DNA.
As used herein, "homologous recombinant animal" refers to an endogenous gene in which the endogenous gene is present in the animal's cells (eg, the animal's embryonic cells) prior to development of the animal. It is a non-human animal, which is altered by homologous recombination with the introduced exogenous DNA molecule, preferably a mammal, and more preferably a mouse.

The transgenic animals of the invention can be introduced into the female pronucleus of fertilized oocytes by, for example, microinjection, retroviral infection, and in pseudopregnant female foster animals. It can be produced by developing an oocyte. This sequence can be introduced as a transgene into the genome of non-human animals. Intron sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. Tissue-specific regulatory sequences can be operably linked to the transgene to direct expression of the polypeptide in particular cells. Methods for producing transgenic animals, particularly animals such as mice, via embryo manipulation and microinjection are conventional in the art and are described, for example, in US Pat. No. 4,736,866. And the same 4,870,
009, U.S. Pat. No. 4,873,191 and Hogan, Manipu.
rating the Mouse Embryo (Cold Spring)
Harbor Laboratory Press, Cold Spring
Harbor, N.M. Y. , 1986). Similar methods are used for the production of other transgenic animals. Founding of transgenic
An animal can be identified based on the presence of the transgene in its genome and / or the expression of mRNA in tissues or cells of the animal. The transgenic founder animal can then be used to breed additional animals carrying the transgene. In addition, transgenic animals carrying a transgene encoding that transgene can be further bred with other transgenic animals carrying other transgenes.

Homologous recombinant host cells may also be produced which allow for in situ alteration of the endogenous polynucleotide sequences of the invention in the host cell genome. The host cells include, but are not limited to, stable cell lines, cells in vivo, or cloned microorganisms. This technology is based on WO93 / 09222 and WO9.
Fully disclosed by 1/12650, WO 91/06667, US Pat. No. 5,272,071 and US Pat. No. 5,641,670. Briefly, a particular polynucleotide sequence corresponding to the polynucleotide or a sequence proximal or distal to the gene is enabled to integrate into the host cell genome by homologous recombination, where expression of the gene can be affected. In one embodiment, regulatory sequences are introduced that either increase or decrease expression of endogenous sequences. Therefore, the protein
It can be produced in cells that normally do not produce the protein. Alternatively, increased expression of the protein may result in cells that normally produce the protein at a particular level. Furthermore, expression can be reduced or eliminated by introducing specific regulatory sequences. The regulatory sequence may be heterologous to the protein sequence, or it may be a homologous sequence with the desired mutation affecting expression. Alternatively, the entire gene can be deleted. The regulatory sequence may be host cell specific or may be capable of functioning in more than one cell type. Still further, certain mutations can be introduced into any desired region of the gene to produce the mutant protein of the invention. Such mutations can be introduced, for example, in specific functional regions.

To produce a homologous recombinant animal, at least a portion of the nucleic acid of the invention, in which a deletion, addition or substitution has been introduced, thereby altering (eg, functionally disrupting) the endogenous gene. A vector containing is prepared. In one embodiment, the vector is designed such that upon homologous recombination its endogenous gene is functionally disrupted (ie, no longer encodes a functional protein) (also referred to as a “knockout” vector). be called). Alternatively, the vector may be designed such that upon homologous recombination, the endogenous gene is mutated or altered but still encodes a functional protein (e.g., the upstream regulatory region directs expression of the endogenous protein). Can be changed to change). In this homologous recombination vector, the altered part of the gene is flanked by additional nucleic acid of the gene at its 5'and 3'ends, whereby the exogenous gene carried by the vector and embryonic stem cells Allows homologous recombination to occur with the endogenous gene for. This additional flanking nucleic acid is long enough for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5'and 3'ends) are included in the vector (eg, for a description of homologous recombination vectors, Thomas and Capecchi (1987) Cell 51: 503). This vector is introduced into embryonic stem cells (eg, by electroporation), and cells in which the introduced nucleic acid has undergone homologous recombination with an endogenous gene are selected (
See, eg, Li et al. (1992) Cell 69: 915). Then
Selected cells can be injected into blastocysts of animals (eg, mice) to form aggregated chimeras (eg, Bradley, Teratocarcinomas).
and Embryonic Stem Cells: A Practica
l Approach, Robertson ed. (IRL, Oxford, 198)
7) See pages 113-152). The chimeric embryo can then be transferred into a suitable pseudopregnant foster animal and the embryo born. Progeny that carry the homologous recombinant DNA in their germ cells can be used to breed animals in which all cells of the animal contain homologous recombinant DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombination animals include
Bradley (1991) Current Opinion in Bio /
Technology 2: 823-829 and PCT Publication WO 90/1.
1354, WO91 / 01140, WO92 / 02968, and WO93 / 0
4169.

In another embodiment, transgenic non-human animals can be produced that contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre / loxP recombinase system of bacteriophage P1. For a description of the cre / loxP recombinase system, see, eg, Lakso et al. (1992) Proc. Natl. Acad. Sci. USA 89: 6232.
See ~ 6236. Another example of a recombinase system is Saccharomy
ces cerevisiae FLP recombinase system (O'Gorm
an et al. (1991) Science 251: 1351-1355). cre /
If the loxP recombinase system is used to regulate expression of the transgene, an animal containing the transgene encoding both the Cre recombinase and the selected protein is needed. Such animals include, for example, two transgenic animals, one containing the transgene encoding the selected protein and the other containing the transgene encoding the recombinase.
Can be provided through the construction of "double" transgenic animals by crossing.

The clones of the non-human transgenic animals described herein also contain W
ilmut et al. (1997) Nature 385: 810-813 and PCT.
It can be made according to the methods described in publications WO 97/07668 and WO 97/07669.

III. Polypeptides The present invention also relates to isolated polypeptides and their encoded by the nucleic acid molecules of the present invention (especially as set forth in SEQ ID NOS: 1-6, 8 and 10). Variants and fragments are provided. For example, as described above, the nucleotide sequence can be used to design a primer for cloning and expressing a cDNA encoding a polypeptide of the invention. Further, the nucleotide sequences of the present invention (eg, the sequences set forth in SEQ ID NOs: 1-6, 8 and 10) are routinely searched by conventional search algorithms (eg, BLAST, Altshul et al. (1990) J. Mo.
l. Biol. 215: 403-410; BLAZE, Brutlag et al. (19
93) Comp. Chem. 17: 203-207) can be used to identify open reading frames (ORFs).

As used herein, a chemical precursor when it is substantially free of cellular material when isolated from recombinant and non-recombinant cells, or when chemically synthesized. Or in the absence of other chemicals, the polypeptide is "isolated"
Or it is said to be "purified". However, a polypeptide may be bound to another polypeptide that normally does not bind in cells, and may still be "isolated" or "purified".

The polypeptide of the present invention can be purified to homogeneity. However, it is understood that preparations in which the polypeptide is not uniformly purified are useful and are considered to include isolated forms of the polypeptide. The important feature is that the preparation allows the desired function of the polypeptide, even in the presence of significant amounts of other components. Thus, the present invention encompasses varying degrees of purity. In one embodiment, the phrase "substantially free of cellular material" refers to less than about 30% (by dry weight) other proteins (ie, contaminating proteins), less than about 20% other proteins, about 10%. Less than other proteins, or less than about 5% other proteins, including preparations of polypeptides.

If the polypeptide is produced recombinantly, it can also be substantially free of culture medium. That is, the culture medium is approximately 20% of the volume of the protein preparation.
Less than, less than about 10%, or less than about 5%. The phrase "substantially free of chemical precursors or other chemicals" includes preparations of polypeptides that are separated from the chemical precursors or other chemicals involved in its synthesis. In one embodiment, the phrase "substantially free of chemical precursors or other compounds" refers to less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical. Precursor or other chemical, less than about 10% chemical precursor or other chemical,
Included are preparations of polypeptides having less than about 5% chemical precursors or other chemicals.

In one embodiment, the polypeptide has an amino acid sequence encoded by a nucleic acid comprising a nucleotide sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 1-6, 8 and 10 and complements thereof. Including. However, the invention also includes sequence variants. Variants include substantially homologous proteins (ie, allelic variants) encoded by the same locus in an organism. The variant also
Substantially to a polypeptide encoded by a nucleic acid which is derived from another locus of the organism but which contains a nucleotide sequence selected from the group consisting of the sequences shown in SEQ ID NOs: 1 to 6, 8 and 10 and their complements. It also includes proteins with high homology. Variants also include proteins that are substantially homologous to these polypeptides but are derived from another organism (ie, orthologs). Variants also include proteins that are substantially homologous to these polypeptides produced by chemical synthesis. Variants also include proteins that are substantially homologous or identical to these polypeptides produced by recombinant methods.

As used herein, two proteins (or regions of those proteins) are at least about 45-55%, typically at least about 70-75%, more representative in amino acid sequence. Typically at least about 80-85%, and most typically at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%.
, 97%, 98%, 99% or more are substantially homologous or identical. Substantially homologous amino acid sequences in accordance with the present invention are nucleic acid sequences selected from the group consisting of the sequences set forth in SEQ ID NOs: 1-6, 8 and 10, or portions thereof, under stringent conditions more often described above. Underneath, it is encoded by a hybridizing nucleic acid.

To determine the percent similarity or identity of two amino acid sequences or two nucleic acids, this sequence is aligned for optimal comparison purposes (eg, optimal alignment with another protein or nucleic acid). For which a gap may be introduced in the sequence of one protein or nucleic acid). The amino acid residues or nucleotides are then compared at corresponding amino acid positions or corresponding nucleotide positions. If a position in one sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the other, then the molecules are homologous at that position. As used herein, amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity." The percent homology between two sequences is a function of the number of identical positions shared by the sequences (ie, the percent homology is
(Number of identical positions / total number of positions) x 100).

The invention also includes polypeptides that have a lower degree of identity, but are sufficiently similar to perform one or more of the same functions performed by the polypeptides encoded by the nucleic acids of the invention. . Similarity is determined by conservative amino acid substitutions. Such a substitution replaces a given amino acid of the polypeptide with another amino acid of similar characteristics. Conservative substitutions are likely to be phenotypically silent. Typically, conservative substitutions are found with the aliphatic amino acid Ala.
, Val, Leu, and Ile with each other; Se at the hydroxyl residue
Exchange between r and Thr, exchange of acidic residues between Asp and Glu, substitution of amide residues between Asn and Gln, exchange of basic residues between Lys and Arg, and aromatic residues Phe, Tyr. It is a replacement between. For guidance on which amino acid changes appear to be phenotypically silent, see Bowie et al. (1990).
Science 247: 1306-1310.

[0081]   (Table 1. Conservative amino acid substitution)

[0082]

[Table 1] Both identity and similarity can be easily calculated (Computation
al Molecular Biology, Lesk, A .; M. Hen, Oxfo
rd University Press, New York, 1988; Bi
ocomputing: Informations and Genome Pr
objects, Smith, D.M. W. Hen, Academic Press, Ne
w York, 1993; Computer Analysis of Seq.
uence Data, Part 1, Griffin, A .; M. And Gri
ffin, H.F. G. Hen, Humana Press, New Jersey, 1
994; Sequence Analysis in Molecular B
iology, von Heinje, G .; , Academic Press,
1987; and Sequence Analysis Primer, Gr.
ibskov, M .; And Devereux, J .; Hen, M Stockton
Press, New York, 1991).

Preferred computer program methods for determining identity and similarity between two sequences include the GCG program package (Devereux, J.
(1984) Nucleic Acids Res. 12 (1): 387),
BLASTP, BLASTN, FASTA (Atschul, SF, et al. (19
90) J. Mol. Biol. 215: 403).

Variant polypeptides include one or more substitutions, deletions, insertions, inversions, fusions and truncations,
Or, any combination of these may differ in amino acid sequence. Furthermore, variant polypeptides can be fully functional or can be deficient in one or more activities. Fully functional variants typically include only conservative modifications or alterations at non-critical residues or non-critical regions. Functional variants can also include substitutions of similar amino acids, which results in no or insignificant changes in function. Alternatively, such substitutions can, to some extent, positively or negatively affect function.

Non-functional variants are typically substitutions, deletions, insertions, inversions or truncations of one or more non-conservative amino acids, or substitutions, insertions, inversions at key residues or regions of interest. Or includes a deletion.

As shown, variants can be naturally occurring or can be made by recombinant means or chemical synthesis so as to provide useful and novel properties of the polypeptide. This includes preventing immunogenicity from the pharmaceutical formulation by preventing protein aggregation.

Amino acids essential for function are determined by methods known in the art (eg, site-directed mutagenesis or alanine scanning mutagenesis (Cunningham et al. (1985)).
Science 244: 1081-1085). The latter procedure introduces one alanine mutation for each residue in the molecule. The resulting mutant molecule is then tested in vitro for biological activity or for proliferative activity in vitro. Sites important for polypeptide activity also include crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al. (1992) J. Mol. Biol. 22).
4: 899-904; de Vos et al. (1992) Science 255: 3.
06-312).

The present invention also includes polypeptide fragments of the polypeptides of the present invention. Fragments can be derived from a polypeptide encoded by a nucleic acid that comprises a nucleotide sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 1-6, 8 and 10 and complements thereof. However, the invention also includes fragments of variants of the polypeptides described herein.

As used herein, a fragment comprises at least 6 consecutive amino acids. Useful fragments include those that retain one or more of the biological activities of the polypeptide, as well as fragments that can be used as immunogens to raise polypeptide-specific antibodies.

Biologically active fragments (eg 6, 9, 12, 15, 20, 30,
A peptide that is 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length is a domain, segment or motif (eg, a peptide, identified by analysis of the polypeptide sequence using well known methods). , Signal peptide,
Extracellular domains, one or more transmembrane segments or loops, ligand binding regions, zinc finger domains, DNA binding domains, acylation sites, glycosylation sites, or phosphorylation sites).

The present invention also provides fragments that have immunogenic properties. These include the epitope-bearing portion of the polypeptides and variants of the invention. These epitope-bearing peptides are useful for raising antibodies that specifically bind to the polypeptide or region or fragment. At least 6 of these peptides
Amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 12 amino acids, at least 1
It can include 4 amino acids, or at least about 15 amino acids to about 30 amino acids. Epitope-bearing peptides or epitope-bearing polypeptides can be obtained by any conventional means (Houghten, RA (1985) Proc. Natl. Acad. Sc).
i. USA 82: 5131-5135). Multiple peptide simultaneous synthesis is described in US Pat. No. 4,631,211.

Fragments can be separate (not fused to another amino acid or polypeptide) or can be within a larger polypeptide. Furthermore, some fragments may be contained within a single, larger polypeptide. In one embodiment, a fragment designed for expression in a host comprises a heterologous prepolypeptide region or propolypeptide region fused to the amino terminus of the polypeptide fragment, and a further fragment fused to the carboxyl terminus of the fragment. May have regions.

Accordingly, the present invention provides chimeric or fusion proteins. These include polypeptides of the invention operably linked to a heterologous protein having an amino acid sequence that is not substantially homologous to the polypeptide. "Operably linked" indicates that the polypeptide protein and the heterologous protein are fused in frame. The heterologous protein can be fused to the N-terminus or C-terminus of the polypeptide. In one embodiment, the fusion protein does not affect the polypeptide function itself. For example, the fusion protein can be a GST fusion protein, where the polypeptide sequence is fused to the N-terminus or C-terminus of the GST sequence. Other types of fusion proteins include enzyme fusion proteins (eg β-galactosidase fusions), yeast two-hybrid GAL.
Fusions, poly-His fusions and Ig fusions are included, but are not limited to. Such fusion proteins, especially poly-His fusions, may facilitate the purification of recombinant polypeptides. In certain host cells, such as mammalian cells, protein expression and / or secretion can be increased using heterologous signal sequences. Thus, in another embodiment, these fusion proteins have the N
Contains a heterologous signal sequence at the end.

EP-A-0 464 533 discloses fusion proteins comprising various portions of immunoglobulin constant regions. Fc is useful in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232 2).
62). For example, in drug development, human proteins have been fused with Fc moieties for the purpose of high throughput screening assays to identify antagonists.
Bennett et al. (1995) Journal of Molecular R
ecognition 8: 52-58 and Johnson et al. (1995).
The Journal of Biological Chemistry
270: 9459-9471. Accordingly, the present invention also includes soluble fusion proteins containing the polypeptides of the present invention and various portions of the constant regions of the heavy or light chains of immunoglobulins (IgG, IgM, IgA, IgE) of various subclasses. To do. The constant part of the heavy chain of human IgG (especially IgG1), where the fusion occurs in the hinge region, is preferred as the immunoglobulin. For some uses, it is desirable to remove Fc after the fusion protein has been used for its intended purpose (eg, when the fusion protein is used as an antigen for immunization). In certain embodiments, the Fc portion may be removed in a simple manner with a cleavage sequence, which also may be incorporated and cleaved with Factor Xa.

Chimeric or fusion proteins can be produced by standard recombinant DNA techniques. For example, DNA fragments encoding different protein sequences are ligated together in frame according to conventional techniques. In another embodiment, the fusion gene may be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of the nucleic acid fragment can be performed using an anchor primer, which produces a complementary overhang between two consecutive nucleic acid fragments, which is subsequently annealed and reamplified to produce the chimeric nucleic acid. Sequences can be generated (Ausubel et al., Current Protocols in
See Molecular Biology, 1992). Furthermore, many expression vectors that already encode a fusion moiety (eg, GST protein) are commercially available. Nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the polypeptide protein.

The isolated polypeptide may be purified from cells which naturally express the polypeptide, cells which have been modified to express it (recombinant), or known polypeptides. It can be synthesized using protein synthesis methods.

In one embodiment, the protein is produced by recombinant DNA technology. For example, a nucleic acid molecule encoding the polypeptide is cloned into an expression vector, the expression vector is introduced into a host cell, and the protein is expressed in the host cell. The protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques.

Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids. In addition, many amino acids, including terminal amino acids, can be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. The routine modifications that occur naturally in polypeptides are described in basic texts, detailed papers and research literature, and these are well known to those skilled in the art.

Thus, the polypeptide may also be such that the amino acid residue that is replaced is not an amino acid residue encoded by the genetic code, that it contains a substituent, or that the mature polypeptide is another compound (eg, that compound). Compounds that increase the half-life of a polypeptide (
A derivative or an additional amino acid (eg, a leader or secretory sequence or a sequence for purification of the mature polypeptide or proprotein sequence) is fused to the mature polypeptide. Includes analog.

Known modifications include acetylation, acylation, ADP-ribosylation, amide formation, covalent attachment of flavins, covalent attachment of heme moieties, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, phosphos. Tidylinositol covalent bond, cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-linking formation, cystine formation, pyroglutamate formation, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxidation, Iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation (
transfer of amino acids to proteins such as arginylation)
Examples include, but are not limited to, NA-mediated addition, and ubiquitination.

Such modifications are well known to those of skill in the art and are described in greater detail in the scientific literature. Some specific general modifications, such as glycosylation, lipid binding, sulfation, gamma-carboxylation of glutamic acid residues, hydoxylation and ADP-ribosylation, are the most basic sources (Proteins-Structure an.
d Molecular Properties, Second Edition, T.D. E. Creig
Hton, W.W. H. Freeman and Company, New Yor
k (1993)). Many detailed reviews are available for this subject (Wold, F., Posttranslational Cov.
alent Modification of Proteins, B.A. B. J
ohnson, ed., Academic Press, New York 1-1
2 (1983); Seifter et al., Meth. Enzymol. 182: 62
6-646 (1990) and Rattan et al. (1992) Ann. N. Y. A
cad. Sci. 663: 48-62).

It is also well known that polypeptides are not always perfectly linear. For example,
Polypeptides may be branched as a result of ubiquitination, and may or may not be generally branched as a result of post-translational events, including natural processing events and events caused by non-naturally occurring human manipulations. However, it can be circular. Cyclic, branched and branched cyclic polypeptides can be synthesized by non-translation natural processes and by synthetic methods.

Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxy termini. Interference with amino or carboxyl groups, or both, in a polypeptide by covalent modifications is common in naturally occurring and synthetic polypeptides. For example, E. coli prior to proteolytic processing. The amino terminal residue of polypeptides produced by E. coli is almost always N-formylmethionine.

The modification can be the action by which the protein is made. For example, for recombinant polypeptides, the modification will be determined by the ability of the host cell to post-translationally modify, and the modification will signal the polypeptide amino acid sequence. Therefore, glycosylation
If desired, the polypeptide is a glycosylated host (generally a eukaryotic cell).
Should be expressed in. Insect cells often perform the same post-translational glycosylation as mammalian cells, and for this reason, insect cell expression systems have evolved to effectively express mammalian proteins with a native pattern of glycosylation.
Similar considerations apply to other modifications.

The same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain more than one type of modification.

Uses of the polypeptides of the present invention are described in detail below. In general, the polypeptides or proteins of the invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods understood in the art. The polypeptides of the present invention can be used to raise antibodies or elicit an immune response. Polypeptides can also be used in assays in reagents (eg,
Used as a labeling reagent) to quantitatively determine the level of proteins or molecules (eg, receptors or ligands) that bind in the biological fluid. Polypeptides can also be used as markers for tissues in which the corresponding protein is preferentially expressed either during substantial tissue differentiation or in disease states. The polypeptides can be used, for example, to isolate corresponding binding partners (eg, receptors or ligands) in an interaction trap assay and screen for small molecules antagonists or agonists of the peptide or its binding interactions.

(IV. Antibodies) In another aspect, the present invention provides polypeptides and peptide fragments of the present invention (for example, a nucleotide selected from the group consisting of the sequences shown in SEQ ID NOs: 1 to 6, 8 and 10). Antibody having an amino acid encoded by a nucleic acid comprising all or part of the sequence). The term "antibody," as used herein, refers to immunoglobulin molecules and portions of immunologically active immunoglobulin molecules (ie, molecules that contain an antigen binding site that specifically binds an antigen). A molecule that specifically binds to the polypeptide of the present invention is a molecule that binds to the polypeptide or a fragment thereof, but does not refer to other molecules in a sample (eg, biological sample) that naturally contains the polypeptide. Substantially unbound Examples of immunologically active portions of immunoglobulin molecules include F (ab) fragments and F (ab ′) 2 which can be generated by treating the antibody with an enzyme (eg, pepsin).
Examples include fragments. The invention provides polyclonal and monoclonal antibodies that bind to the polypeptides of the invention. The terms "monoclonal antibody" or "monoclonal antibody composition," as used herein, contain only one antigen binding site capable of immunoreacting with a particular epitope of a polypeptide of the invention. Refers to a group of. Thus, a monoclonal antibody composition typically displays a single binding affinity for a particular polypeptide of the invention with which it immunoreacts.

Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with the desired immunogen, eg, a polypeptide of the invention or fragment thereof. Antibody titers in immunized subjects can be monitored over time by standard techniques (eg, using an enzyme linked immunosorbent assay (ELISA) with immobilized polypeptide). If desired, antibody molecules to the polypeptide can be isolated from mammals (eg, from blood) and further purified by well-known techniques (eg, Protein A chromatography to obtain IgG fractions). obtain. At the appropriate time after immunization, for example, when the antibody titer is highest, antibody-producing cells can be obtained from the subject and used to use standard techniques (eg, initially Kohler and Milstein ( 1975) Nature 256
: 495-497, human B cell hybridoma technology (Kozbor et al. (1983) Immunol. Today 4:72.
), EBV hybridoma technology (Cole et al. (1985), Monoclona).
l Antibodies and Cancer Therapy, Alan
R. Liss, Inc. , 77-96) or the trioma.
Technology) to prepare monoclonal antibodies. Techniques for producing hybridomas are well known (generally, Current Protocols in Im.
Munology (1994) Coligan et al. (ed.), John Wiley
& Sons, Inc. , New York, NY). Briefly, immortalized cell lines (typically myeloma cells) are fused with immunogens as described above with immunized lymphocytes (typically spleen cells) of mammalian origin and obtained. Hybridoma cell culture supernatants are screened to identify hybridomas that produce monoclonal antibodies that bind the polypeptides of the invention.

Any of a number of well-known protocols used to fuse lymphocytes with immortalized cell lines can be applied for the purpose of producing monoclonal antibodies against the polypeptides of the present invention (eg, Current Protocols in Imm
Unology, supra; Galfre et al. (1977) Nature 266: 5.
50-52; H. Kenneth, Monoclonal Antibod
ies: A New Dimension In Biological An
alyses, Plenum Publishing Corp. , New Y
ork, New York (1980); and Lerner (1981) Ya.
le J. Biol. Med. , 54: 387-402). Moreover, one skilled in the art will understand that there are many variations of such methods,
These are still useful. Typically, the immortalized cell line (eg, myeloma cell line) is from the same mammalian species as the lymphocytes. For example, a mouse hybridoma is a lymphocyte derived from a mouse immunized with an immunogenic preparation of the present invention and an immortalized mouse cell line (for example, a culture medium containing hypoxanthine, aminopterin and thymidine (
Myeloma cell line) that is sensitive to "HAT medium"). Any of a number of myeloma cell lines can be used as fusion partners according to standard techniques (eg P3-NS1 / 1-Ag4-1, P3-x63-Ag8.
． 653 or Sp2 / O-Ag14 myeloma line). These myeloma lines are ATC
Available from C. Typically, HAT-sensitive mouse myeloma cells are fused to mouse spleen cells using polyethylene glycol ("PEG"). The hybridoma cells obtained from the fusion are then selected using HAT medium, which kills the unfused cells and the non-producing fused myeloma cells (the unfused spleen cells are transformed. Not so die a few days later)). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind a polypeptide of the invention (eg, using a standard ELISA assay).

As an alternative to preparing monoclonal antibody-secreting hybridomas, monoclonal antibodies to the polypeptides of the invention can be identified and recombinant combinatorial immunoglobulin libraries (eg, antibody phage display libraries) screened with the polypeptides. Can be isolated, thereby isolating an immunoglobulin library member that binds the polypeptide.
Kits for generating and screening phage display libraries are commercially available (eg, the Pharmacia Recom).
BINANT PHAGE ANTIBODY SYSTEM, Catalog No. 2
7-9400-01; and the Stratagene Surf ZAPT.
M Phase Display Kit, Catalog No. 240612). Further, examples of methods and reagents that are particularly amenable to use in generating and screening antibody display libraries are found, for example, in US Pat.
409; PCT Publication No. WO92 / 18619; PCT Publication No. WO91 / 1
7271; PCT publication number WO92 / 20791; PCT publication number WO92 / 1
5679; PCT publication number WO93 / 01288; PCT publication number WO92 / 0
1047; PCT publication number WO92 / 09690; PCT publication number WO90 / 0
2809; Fuchs et al. (1991) Bio / Technology 9:13.
70-1372; Hay et al. (1992) Hum. Antibod. Hybrid
mas 3: 81-85; Huse et al. (1989) Science 246:
1275-1281; Griffiths et al. (1993) EMBO J. 12:
725-734.

Further, recombinant antibodies (eg, chimeric and humanized monoclonal antibodies) that include both human and non-human portions are within the scope of the invention, which uses standard recombinant DNA techniques. Can be made by Such chimeric and humanized monoclonal antibodies can be prepared by recombinant DNA techniques known in the art (eg, PCT
Publication number WO87 / 02671; European patent application 184,187; European patent application 171,496; European patent application 173,494; PCT publication number WO86 /
01533; U.S. Pat. No. 4,816,567; European Patent Application 125,023; Better et al. (1988) Science 240: 1041-1043;
Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:
3439-3443; Liu et al. (1987) J. Am. Immunol. 139: 35
21-3526; Sun et al. (1987) Proc. Natl. Acad. Sci
． USA 84: 214-218; Nishimura et al. (1987) Canc.
． Res. 47: 999-1005; Wood et al. (1985) Nature 3
14: 446-449; and Shaw et al. (1988) J. Am. Natl. Canc
er. Inst. 80: 1553-1559; Morrison (1985) S.
science 229: 1202-1207; Oi et al. (1986) Bio / Te.
chnies 4: 214; US Pat. No. 5,225,539; Jones
(1986) Nature 321: 552-525; Verhoeyan et al. (1988) Science 239: 1534; and Beidler et al. (1).
988) J. Immunol. 141: 4053-4060).

Completely, human antibodies are particularly desirable for therapeutic treatment of human patients. Such antibodies may be produced using transgenic mice that are incapable of expressing the endogenous immunoglobulin heavy and light chain genes but are capable of expressing human heavy and light chain genes. The transgenic mouse is immunized in the normal fashion with a selected antigen (eg, all or part of a polypeptide of the invention). Monoclonal antibodies against this antigen can be obtained using conventional hybridoma technology.
This human immunoglobulin transgene carried by transgenic mice is rearranged during B cell differentiation and subsequently undergoes class switching and somatic mutations. Therefore, using such a technique, therapeutically useful IgG, IgA
And it is possible to generate IgE antibodies. For an overview of this technique for purifying human antibodies, see Lonberg and Huszar (1995, Int.
． Rev. Immunol. 13: 65-93). For a detailed discussion of this technique for producing human antibodies and human monoclonal antibodies, see, eg, US Pat. No. 5,625,126; US Pat. No. 5,633.
, 425; U.S. Pat. No. 5,569,825; U.S. Pat. No. 5,661,016.
And U.S. Patent No. 5,545,806. In addition, Abgen
ix, Inc. Companies such as (Freemont, CA) may ensure that they provide human antibodies against selected antibodies using techniques similar to those described above.

Completely human antibodies recognizing selected epitopes can be generated using the technique referred to as "guided selection." In this approach, selected non-human monoclonal antibodies (eg mouse antibodies) are used to guide the selection of human antibodies that recognize the same epitope entirely.

The use of the antibodies of the present invention is described in detail below. Generally, the antibodies of the invention (
For example, a monoclonal antibody) can be used to isolate a polypeptide of the invention by standard techniques, such as affinity chromatography or immunoprecipitation. Polypeptide-specific antibodies can facilitate the purification of naturally occurring polypeptides from cells and recombinantly produced polypeptides expressed in host cells. In addition, antibodies specific for the polypeptides of the invention can be used to detect the polypeptide (eg, in cell lysates or cell supernatants) to assess the expression level and expression pattern of the polypeptide. Antibodies can be used, for example, diagnostically to monitor protein levels in tissues as part of a clinical trial procedure (eg, to determine the efficacy of a given treatment regimen). Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic groups include streptavidin / biotin and avidin / biotin; suitable Examples of fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine, fluorescein, dansyl chloride or phycoerythrin; examples of luminescent substances include luminol; As the luminescent substance, luciferase,
Luciferin, and aequorin are included, and examples of suitable radioactive materials include ¹²⁵ I, ¹³¹ I, ³⁵ S or ³ H.

V. Computer Readable Methods The nucleotide or amino acid sequences of the invention are also provided in a variety of means to facilitate their use. As used herein, "provided" means that an article of manufacture other than an isolated nucleic acid or amino acid molecule comprises a nucleotide sequence or amino acid sequence of the invention. Such articles of manufacture provide the nucleotide or amino acid sequences, or a subset thereof (open reading frame (ORF)) in some form. This form allows the person skilled in the art to test the nucleotide or amino acid sequences, or a subset thereof, indirectly using suitable means. This is because it is in a naturally occurring or purified form.

In one application of this embodiment, the nucleotide or amino acid sequences of the invention can be recorded on a computer-readable medium. As used herein, "computer-readable medium" means any medium that can be directly read and accessed by a computer. As such a medium,
Magnetic storage medium (floppy (registered trademark) disk, hard disk storage medium,
And tape); optical storage medium (CD-ROM etc.); electrical storage medium (
RAM and ROM); and hybrids of these categories, such as magnetic / optical storage media, but are not limited thereto. One of skill in the art will readily appreciate that any presently known computer readable medium can be used to make an article of manufacture containing the computer readable medium that records the nucleotide or amino acid sequences of the invention on the computer readable medium. Recognize correctly.

As used herein, "recorded" refers to the process of storing information on a computer-readable medium. One of skill in the art can readily employ any of the presently known methods of recording information on computer-readable media to produce an article of manufacture that comprises a nucleotide or amino acid sequence of the invention.

A variety of data storage structures are available to those of skill in the art to make computer-readable media for recording the nucleotide or amino acid sequences of the present invention. The choice of data storage structure is generally based on the means chosen to access the stored information. In addition, a variety of data processors and formats may be used to store the nucleotide sequence information of the present invention on computer readable media. Sequence information may be represented in a word processing text file. This file is created according to the format of commercially available software (such as WordPerfect and Microsoft Word) or AS
CII file (stored in database application (DB2,
(Sybase, Oracle, etc.)) format. One of ordinary skill in the art can readily adapt any number of data processor construction formats (eg, text files or databases) to obtain a computer-readable medium for recording the nucleotide sequence information of the present invention.

By providing the nucleotide or amino acid sequences of the invention in computer readable form, one of skill in the art can routinely access sequence information for a variety of purposes. For example, one of skill in the art can use the nucleotide or amino acid sequences of the invention in computer readable form to compare the sequence information stored in the data storage means to the target sequence or target structural motif. Searching means are used to identify fragments or regions of the sequences of the invention that match a particular target sequence or target motif.

As used herein, a “target sequence” is 6 or more nucleotides or 2
It can be any DNA or amino acid sequence of the above amino acids. Those skilled in the art
It can easily be seen that the longest target sequence, such as the smallest target sequence, exists as random occurrences in the database. The most preferred sequence length of the target sequence is about 10 to about 100 amino acids or about 30 to about 300 nucleotide residues. However, it is well understood that commercially important fragments, such as sequence fragments associated with gene expression and protein processing, can be shorter.

As used herein, "target structure motif" or "target motif"
Refers to any reasonably selected sequence or combination of sequences for which a sequence is selected based on the three-dimensional arrangement formed by the folding of the target motif. There are various target motifs known in the art. Protein target motifs include, but are not limited to, enzyme active sites and signal sequences. Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin constructs and inducible expression elements (protein conjugating enzymes).

Computer software is publicly available to allow those skilled in the art to access sequence information provided on computer-readable media for analysis of other sequences and comparison with other sequences. To do. A variety of known algorithms are publicly disclosed, and a variety of commercially available software to guide the search means are and may be used in the computer-based system of the present invention. Examples of such software include MacPattern (EMBL) and BLAST.
N and BLASTX (NCBIA), but are not limited thereto.

For example, BLAST on the Sybase system (Altschul et al. (199
0) J. Mol. 215: 403-410) and BLAZE (Brutlag.
(1993) Comp. Chem. 17: 203-207) using a software implementing a search algorithm, an open reading frame (O) of the sequence of the invention containing homology to ORFs or proteins from other libraries.
PF) can be identified. Such ORFs are protein-encoding fragments and are useful in the production of commercially important proteins, such as the enzymes used in various reactions, and the production of commercially useful metabolites.

VI. Detection Assay Portions or fragments of the nucleotide sequences (and corresponding complete gene sequences) identified herein can be used in many ways as polynucleotide reagents. For example, these sequences can be used for: (i) to map each gene on a chromosome, thereby locating a genetic region associated with a genetic disease; (ii) minor biological To identify individuals from a sample (tissue typing); and (iii) to aid in forensic identification of biological samples. These applications are described in the subsections below.

1. Chromosome Mapping Once a nucleic acid (or part of a sequence) is isolated, this nucleic acid can be used to map the location of the gene on the chromosome. Mapping sequences to chromosomes is an important first step in relating these sequences to genes associated with disease. Briefly, a gene can be derived from the nucleic acid molecules described herein to PC
It can be mapped to the chromosome by preparing an R primer (preferably 15-25 bp in length). Computer analysis of sequences shows 1 in genomic DNA
It can be used to predict primers that span no more than one exon (and thus complicate the amplification process). These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only hybrids containing the human gene corresponding to the appropriate nucleotide sequence will produce an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells of various mammal origin (eg, human and mouse cells). As the hybrids of human and mouse cells grow and divide, they gradually lose the human chromosomes in a random order, but retain the mouse chromosomes. By using a medium in which mouse cells cannot grow (because it lacks certain enzymes) but human cells can grow, one of the human chromosomes containing the gene encoding the required enzyme is retained. It By using different media, a panel of hybrid cell lines can be established. Each cell line in the panel contains either a single human chromosome or a few human chromosomes, and a complete set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes . (D'Eustachio
(1983) Science 220: 919-924). Somatic cell hybrids containing only fragments of the human chromosome can also be generated by using human chromosomes with translocations and deletions.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning specific sequences to specific chromosomes. More than two sequences can be allocated per day using one thermal cycler. Using the nucleic acid molecules of the invention to design oligonucleotide primers, sublocalization can be achieved with a panel of fragments from a particular chromosome. Other mapping strategies that can also be used to map the defined sequences to that chromosome include in situ hybridization (Fan et al. (1990)
) PNAS 97: 6223-27), prescreening with labeled flow-sorted chromosomes, and preselection by hybridization to a chromosome-specific cDNA library.

Fluorescence in situ hybridization (FISH) of nucleotide sequences to metaphase chromosome expansion can also be used to provide the correct chromosomal location in one step. Chromosome unfolding can be done using cells that are blocked at metaphase by a chemical, such as colcemid, which divides the spindle. Chromosomes can be briefly treated with trypsin and then Giemsa stained. A pattern of light and dark zones develops on each chromosome, which allows the chromosomes to be individually identified. FISH technology can be used with nucleotide sequences as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher binding probability for a particular chromosomal location with sufficient signal strength for simple detection. In order to get good results in a reasonable time, preferably
1,000 bases, and more preferably 2,000 bases are sufficient. For an overview of this technology, see Verma et al. (Human Chromosomes: A
Manual of Basic Technologies (Pergamon)
See Press, New York, (1988)).

Reagents for chromosome mapping can be used to individually mark single chromosomes or single sites on that chromosome. Alternatively, a panel of reagents can be used to mark multiple sites and / or multiple chromosomes. Reagents corresponding to the non-coding regions of this gene are indeed preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, which increases the chance of cross-hybridization during chromosome mapping.

Once the sequence has been mapped to a precise chromosomal location, the physical location of the sequence on the chromosome can be correlated with genetic map data. (Such data is, for example,
V. McKusick, Mendelian Inheritance in
Man (Johns Hopkins University Welch M
(available online through the Medical Library)). The relationship between genes and diseases that map to the same chromosomal region is then described, for example, by Egeland et al. (1987) Nature 325: 783-7.
87, which can be identified through linkage analysis (co-inheritance of physically adjacent genes).

Furthermore, differences in DNA sequences between diseased and unaffected individuals associated with defined genes can be determined. If the mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation may be the causative agent of the particular disease. A comparison of affected and unaffected individuals generally involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible from chromosomal unfolding or are detectable using PCR based on their DNA sequences. Including the step of searching. Finally, complete sequencing of genes from some individuals can be performed to confirm the presence of the mutation and to distinguish the mutation from polymorphisms.

2. Tissue Typing The nucleotide sequences of the present invention can also be used to identify individuals from small biological samples. For example, the United States Armed Forces is considering the use of restriction fragment length polymorphism (RFLP) to identify its staff. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes and probed on a Southern blot to generate a unique band for identification. This method can be lost, replaced, or stolen, making “Dog Tag (Do
g Tag) ”is not subject to the current restrictions. The sequences of the present invention are useful as additional DNA markers for RFLP (described in US Pat. No. 5,272,057).

In addition, the sequences of the present invention provide for the actual base-by-base D of selected portions of the genome of an individual.
It can be used to provide an alternative technique for determining NA sequences. Therefore, the nucleic acid molecules described herein can be used to prepare two PCR primers from the 5'and 3'ends of the sequence. These primers can then be used to amplify the individual's DNA and subsequently sequence it.

A panel of corresponding DNA sequences from an individual prepared in this manner can provide identification of a unique individual. This is because each individual has a unique set of such DNA sequences due to allelic differences. The sequence of the present invention is
It can be used to obtain such identifying sequences from individuals and from tissues. The nucleic acid molecule of the present invention uniquely represents a portion of the human genome. Allelic modifications occur to some extent in the coding regions of these sequences and to a greater extent in non-coding regions. Allelic alterations between individual humans are estimated to occur at a frequency of approximately 1 in 500 bases. Each of the sequences described herein can, to some extent, be used as a standard for DNA from an individual that can be compared for identification purposes. Since more polymorphisms occur in the non-coding regions, fewer sequences are needed to distinguish individuals. The non-coding sequences of these sequences may be sufficient to provide positive individual identification with a panel of perhaps 10-1,000 primers, each yielding a 100 base non-coding amplified sequence. Putative coding sequence (eg, SEQ ID NO: 3, SEQ ID NO: 19,
When SEQ ID NO: 31 or SEQ ID NO: 43) is used, a more suitable number of primers for positive individual identification is 500-2,000.

When the panel of reagents from the nucleic acid molecules described herein is used to generate a specific identification database for an individual, these same reagents are:
It can later be used to identify tissue from that individual. Using a specific identification database, positive identification of individuals (live or dead) can be made from very few tissue samples.

3. Use of Partial Nucleic Acid Molecular Sequences in Forensic Biology DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific discipline that uses genotyping of biological evidence found at the scene of an incident, for example, as a means to positively identify perpetrators of crime. To make such identifications, PCR technology has been found in organizations found in crime scenes (eg,
It can be used to amplify DNA sequences taken from very small biological samples such as hair or skin) or body fluids (eg blood, saliva or semen). The amplified sequence can then be compared to a standard, which allows identification of the origin of the biological sample.

The sequences of the invention can be used to provide polynucleotide reagents (eg, PCR primers) targeted to specific loci in the human genome, eg, another “identifying marker”. By providing (ie, another DNA sequence that is unique to a particular individual), the reliability of DNA-based forensic identification can be enhanced. As mentioned above, the actual nucleotide sequence information can be used for identification as an accurate alternative to the pattern formed by the restriction enzyme generated fragments. Sequences targeted to the non-coding regions of the sequences described herein are particularly suitable for this application if a greater number of polymorphisms occur in this non-coding region. This is
This technique makes it easy to distinguish individuals. Examples of polynucleotide reagents include nucleic acid molecules of the invention or portions thereof (eg, fragments having a length of at least 20 bases, preferably at least 30 bases).

The nucleic acid molecules described herein further include polynucleotide reagents, such as labeled or labelable probes, which can be used, for example, in in situ hybridization techniques to identify specific tissues. )
Can be used to provide This can be very useful when a forensic pathologist is presented with tissue of unknown origin. Such a panel of probes can be used to identify tissue by species type and / or organ type.

In a similar fashion, these reagents, primers or probes can be used to screen tissue cultures for contaminants (ie, for the presence of a mixture of different cell types in culture).

VII. Predictive Medicine: The present invention also relates to the field of predictive medicine in which diagnostic assays, prognostic assays, and clinical trial monitoring are used for prognostic (predictive) purposes, thereby prophylactically assessing an individual. Treat. Accordingly, one aspect of the invention relates to diagnostic assays for determining the expression and protein activity of the proteins and / or nucleic acids of the invention in the context of biological samples (eg blood, serum, cells, tissues). , Thereby determining whether the individual suffers from or is at risk of developing a disease or disorder associated with aberrant expression or activity. The invention also provides a prognostic (or predictive) assay for determining whether an individual is at risk of developing a disorder associated with the activity or expression of a protein or nucleic acid of the invention.

Disorders associated with programmed cell death are particularly relevant, as discussed in detail herein below.

For example, mutations in a specified gene can be assayed in the biological sample. Such assays may be used for prognostic or prophylactic purposes, whereby individuals are characterized by the expression or activity of the nucleic acid molecules or proteins of the invention, or prior to the onset of disorders associated therewith. Can be treated prophylactically.

Another aspect of the invention relates to monitoring the effect of factors (eg drugs, compounds) on the expression or activity of the proteins of the invention in clinical trials.

[0144]   These and other factors are described in further detail in the sections below.

1. Diagnostic Assays An exemplary method for detecting the presence or absence of a protein or nucleic acid of the invention in a biological sample is to obtain a biological sample from a test subject, And a biological sample with the protein or nucleic acid (eg, mR
NA, genomic DNA), which encodes the protein, is contacted with a detectable compound or agent, thereby detecting the presence of the protein or nucleic acid in the biological sample. A preferred agent for detecting mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to the mRNA or genomic DNA described herein. Nucleic acid probes are, for example, full length nucleic acids or portions thereof (eg, at least 15, 30, 50, 100, 250).
Or an oligonucleotide of length 500 nucleotides, and an oligonucleotide sufficient to specifically hybridize to the appropriate mRNA or genomic DNA under stringent conditions). For example, the nucleic acid probe is SEQ ID NO: 1.
˜6, 8 and 10 all or part of the sequence shown, or SEQ ID NO: 1
It may be the complement of the sequences shown in ˜6, 8 and 10 or parts thereof. Other probes suitable for use in the diagnostic assays herein are described herein.

In one embodiment, the preferred agent for detecting a protein of the invention is an antibody capable of binding to this protein, preferably an antibody with a detectable label.
Is. The antibody can be a polyclonal antibody, or more preferably a monoclonal antibody. Intact antibodies or fragments thereof (eg Fab or F (ab ′) ₂ ) can be used. With respect to a probe or antibody, the term "labeled" refers to the direct labeling of a probe or antibody by the binding of a detectable substance to the probe or antibody (ie, physical linkage), as well as to another reagent that is directly labeled. It is intended to include indirect labeling of the probe or antibody with the reaction. Examples of indirect labeling include detection of primary antibody using a fluorescently labeled secondary antibody,
And end labeling of the DNA probe with biotin, which allows it to be detected with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present in a subject. That is,
The detection method of the invention can be used to detect the mRNA, protein or genomic DNA of the invention in a biological sample in vitro and in vivo. For example, in vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detecting proteins include solid-phase enzyme-linked immunosorbent assay (ELI).
SA), Western blot, immunoprecipitation and immunofluorescence. In vitro techniques for detection of genomic DNA include Southern hybridizations. In addition, in vivo techniques for detection of proteins include the step of introducing a labeled protein antibody into the subject. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

In one embodiment, the biological sample comprises protein molecules from the test subject. Alternatively, the biological sample may contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a serum sample or biopsy isolated by conventional means from a subject.

In another embodiment, the method further comprises the steps of: obtaining a control biological sample from a control subject, the control sample being provided with a protein, mRNA, or genomic DNA of the invention. Contact with a detectable compound or agent, resulting in protein, mRNA, or genomic D
NA is detected in the biological sample, and comparing the presence of protein, mRNA, or genomic DNA in the control sample to the presence of protein, mRNA, or genomic DNA in the test sample.

The invention also includes a kit for detecting the presence of a protein or nucleic acid of the invention in a biological sample (test sample). For example, the kit comprises a labeled compound or agent capable of detecting a protein or mRNA in a biological sample; and means for determining the amount in the sample; and comparing the amount in the sample to a standard. Means for doing so may be included. This compound or drug
It can be packaged in a suitable container. The kit can further include instructions for using the kit to detect proteins or nucleic acids.

2. Prognostic Assay The diagnostic methods described herein further include having or at risk of developing a disease or disorder associated with aberrant expression or activity of the proteins and nucleic acids of the invention. It can be used to identify sexual subjects. Therefore, the term "diagnosis"
The term not only refers to ascertaining whether a subject has an active disease,
It relates to ascertaining whether a subject is predisposed to developing an active disease, and to determine the likelihood that treatment of an active disease will be effective. For example, an assay described herein (eg, the diagnostic assay described above or the assay below) may be performed using disorders associated with protein or nucleic acid expression or activity (eg, proliferative, differentiation or developmental disorders, or hematopoietic disorders). ), Or at risk of developing it. Alternatively, prognostic assays can be utilized to identify subjects who have or are at risk of developing a differentiation or proliferative disorder (eg, cancer). Accordingly, the invention provides a method for identifying a disease or disorder associated with aberrant expression or activity of a protein or nucleic acid molecule of the invention, wherein a test sample is obtained from the subject and the protein or Nucleic acid (eg, mRNA, genomic DNA) is detected, where the presence of the protein or nucleic acid has or develops a disease or disorder associated with aberrant expression or activity of the protein or nucleic acid sequences of the invention. A diagnosis of a subject at risk. As used herein, "test sample" refers to a biological sample obtained from a subject of interest.
For example, the test sample can be a sample of biological fluid (eg, serum), cells or tissues.

Disorders associated with programmed cell death are of particular relevance, as discussed in detail herein below.

In addition, the prognostic assay used herein refers to an agent (eg, agonist, drug) for treating a disease or disorder in which a subject is associated with aberrant expression or activity of a protein or nucleic acid molecule of the invention. Antagonists, peptidomimetics, proteins, peptides, nucleic acids, small molecules, or other drug candidates) can be administered. For example, such a method may be used to
For example, it can be used to determine whether it can be effectively treated with a particular agent for a proliferative disorder, a differentiation disorder or a developmental disorder). Alternatively, such methods can be used to determine whether a subject can be effectively treated with an agent for a differentiation or proliferative disorder (eg, cancer). Therefore, the present invention provides that the subject is
Providing a method for determining whether it can be effectively treated with an agent for a disorder associated with aberrant expression or activity of a protein or nucleic acid molecule of the invention,
Here, a test sample is obtained and the expression or activity of the protein or nucleic acid is detected (eg, where the abundance of expression or activity of a particular protein or nucleic acid is a disorder associated with aberrant expression or activity). Is a diagnosis for a subject that may be administered a drug to treat.

Disorders associated with programmed cell death are particularly relevant, as discussed in detail herein below.

The methods of the invention can also be used to detect genetic mutations in a gene or nucleic acid molecule of the invention, whereby a subject carrying a mutated gene undergoes abnormal development, abnormal cell differentiation, Determine whether at risk for injury characterized by abnormal cell proliferation or abnormal hematopoietic response. In a particular embodiment, the method provides for the mutation of a gene in a sample of cells from a subject characterized by the integrity of a gene encoding a particular protein or at least one alteration affecting misexpression of this gene. Detecting the presence or absence. For example, such genetic mutations can be detected by confirming the presence of at least one of the following: (1) deletion of one or more nucleotides; (2
) Addition of one or more nucleotides; (3) Substitution of one or more nucleotides; (4
) Chromosomal rearrangements; (5) Changes in the level of messenger RNA transcripts; (
6) aberrant modifications such as methylation patterns of genomic DNA; (7) presence of non-wild type splicing patterns of messenger RNA transcripts; (8) non-wild type levels; (9) allelic deletion; and (10). Improper post-translational modification. As described herein, there are numerous assay techniques known in the art that can be used to detect mutations in particular genes. A preferred biological sample is
A tissue or serum sample isolated by conventional means from a subject.

In a particular embodiment, the detection of this mutation is performed by polymerase chain reaction (PCR
) (See, eg, US Pat. Nos. 4,683,195 and 4,683,202) (eg, anchor PCR or RACE PCR),
Alternatively, a ligated chain reaction (LCR) (see, eg, Landegran et al., (1998)
) Science 241: 1077-1080; and Nakazawa et al.,
(1994) PNAS 91: 360-364), the latter of which may be particularly useful for detecting point mutations (eg, Abravaya et al., (1995) Nucleic. Acid
s Res. 23: 675-682). The method includes the following steps: collecting a sample of cells from a patient, nucleic acid (eg, genomic, m
RNA, or both) from the cells of this sample, one or more primers that specifically hybridize to the gene under conditions such that hybridization and amplification of the gene (if any) occurs. Contacting a nucleic acid sample, and detecting the presence or absence of an amplification product or detecting the size of this amplification product and comparing its length with a control sample. It is anticipated that PCR and / or LCR may be desirable to use as the primary amplification step in combination with any of the techniques used to detect the mutations described herein.

Alternative amplification methods include: self-sustaining sequence replication (self s).
used sequence replication) (Guate
Ili, J .; C. Et al. (1990) Proc. Natl. Acad. Sci. U
SA 87: 1874-1878), transcription amplification system (transcription)
al amplification system) (Kwoh et al., (1989)
) Proc. Natl. Acad. Sci. USA 86: 1173-1177
), Q-β replicase (Lizardi et al., (1988) Bio / Techn.
6: 1197), or any other method of nucleic acid amplification, followed by detection of the amplified molecule using techniques well known to those of skill in the art. These detection schemes are particularly useful for detecting nucleic acid molecules when such nucleic acid molecules are present in very low numbers.

In an alternative embodiment, mutations of a given gene from a sample cell can be identified by alterations in the restriction enzyme cleavage pattern. For example, sample DNA and control DNA are isolated, amplified (optionally), digested with one or more restriction endonucleases, and the size of the fragment length determined by gel electrophoresis. And compared. Differences in fragment length size between sample DNA and control DNA are due to sample D
Shows mutations in NA. In addition, the use of sequence-specific ribozymes (see, eg, US Pat. No. 5,498,531) can be used to count for the presence of particular mutations due to the development or loss of ribozyme cleavage sites.

In other embodiments, genetic mutations can be identified by hybridizing sample and control nucleic acids (eg, DNA or RNA) to high density arrays containing hundreds or thousands of oligonucleotide probes. (
Cronin et al. (1996) Human Mutation 7: 244-2.
55; Kozal et al. (1996) Nature Medicine 2:75.
3-759). For example, genetic mutations are described in Cronin, M .; T. Can be identified in a two-dimensional array containing photogenerated DNA probes as described by et al., Supra. Briefly, a first hybridization array of probes can be used to identify base changes between sequences by scanning through long stretches of DNA in samples and controls to create a linear array of consecutive overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of the particular mutation by using a smaller, specialized probe array complementary to all detected modifications or mutations. Done. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactions known to those of skill in the art may be used to directly sequence a gene and the gene sequence of this sample to the corresponding wild type (control). Mutations can be detected by comparison with the gene sequence. An example of a sequencing reaction is Maxim and Gilbert ((1977
) PNAS 74: 560) or Sanger ((1977) PNAS 74
: 5463). Any of a variety of automated sequencing procedures can be performed by mass spectrometry sequencing (eg, PCT Publication No. WO9.
4/16101; Chosen et al. (1996) Adv. Chromatogr
． 36: 127-162; and Griffin et al., (1993) Appl. B
iochem. Biotechnol. 38: 147-159), diagnostic assays (1995) Biotechniques 19: 448).
It is considered that it can be utilized when performing.

As another method for detecting mutations, protection from cleaving agents is RNA / RN
The methods used to detect mismatched bases in A or RNA / DNA heteroduplexes are included (Myers et al. (1985) Science 23).
0: 1242). Generally, the technique of "mismatch cleavage" is formed by hybridizing (labeled) RNA or DNA containing wild-type sequences with potentially mutated RNA or DNA obtained from a tissue sample. Start by providing a heteroduplex. Double-stranded duplexes are treated with an agent that cleaves the single-stranded region of the duplex such that it is present due to a base pair mismatch between the control and sample strands. For example, RNA / DNA duplexes can be treated with RNase, which digests mismatched regions, and DNA / DNA hybrids can be treated with S1 nuclease, which enzymatically digests mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on a denaturing polyacrylamide gel to determine the site of mutation. For example, Co
tton et al., (1988) Proc. Natl. Acad. Sci. USA 8
5: 4397; Saleeba et al., (1992) Methods Enzymo.
l. 217: 286-295. In certain embodiments, control DNA or RNA can be labeled for detection.

In yet another embodiment, the mismatch cleavage reaction recognizes mismatched base pairs in double-stranded DNA in a defined system for detecting and mapping point mutations in cDNA obtained from a sample of cells. One or more proteins (so-called "DNA mismatch repair" enzymes) that do this. For example, E. E. coli mutY enzyme cleaves A at a G / A mismatch and He
Thymidine DNA glycosylase from La cells cleaves T at a G / T mismatch (Hsu et al., (1994) Carcinogenesis 15: 1657.
-1662). According to an exemplary embodiment, the nucleotide sequence-based probes of the present invention are hybridized to cDNA or other DNA products from test cells. The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols and the like. See, for example, US Pat. No. 5,459,039).

In another embodiment, alterations in electrophoretic mobility are used to identify mutations in genes. For example, single-stranded conformational polymorphism (SSCP)
Can be used to detect differences in electrophoretic mobility between mutant and wild-type nucleic acids (Orita et al. (1989) Proc. Natl. Acad. Sc).
i. USA 86: 2766; Cotton (1993) Mutat. Res.
285: 125-144; and Hayashi (1992) Genet. An
al. Tech. Appl. See also 9: 73-79). Single-stranded DNA fragments of sample nucleic acid and control nucleic acid are denatured and allowed to regenerate. The secondary structure of single-stranded nucleic acids varies according to sequence, and the resulting changes in electrophoretic mobility allow even single base changes to be detected. DN
The A fragment can be labeled or detected with a labeled probe. The sensitivity of the assay can be enhanced by using RNA (rather than DNA), where secondary structure is more sensitive to changes in sequence.
In one embodiment, the method utilizes heteroduplex analysis to separate double-stranded heteroduplex molecules based on changes in electrophoretic mobility (Keen et al.,
(1991) Trends Genet. 7: 5).

In yet another embodiment, the movement of the mutant or wild-type fragment in polyacrylamide containing a denaturing agent gradient is performed by denaturing gradient gel electrophoresis (DGGE).
(Myers et al., (1985) Nature 313.
: 495). When DGGE is used as a method of analysis, the DNA is modified to ensure that it is not completely denatured, for example by adding a GC clamp of high melting GC rich DNA of about 40 bp by PCR. . In a further embodiment, a temperature gradient is used instead of a denaturing gradient to identify differences in the mobility of control and sample DNA (Ros.
enbaum and Reissner (1987) Biophys. Chem.
265: 12753).

Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers can be prepared in which known mutations are centrally replaced and then hybridized to the target DNA under conditions that allow hybridization only if a perfect match is found (Saiki). Et al. (1986) Nature 324: 163; Sa.
iki et al. (1989) Proc. Natl. Acad. Sci. USA 86
: 6230). Such an allele-specific oligonucleotide is attached to the membrane to which the oligonucleotide hybridizes and labeled with the target DN.
When hybridized with A, it hybridizes to the PCR amplified target DNA or a number of different mutations.

Alternatively, allelec-specific amplification techniques that rely on selective PCR amplification can be used in combination with the present invention. Oligonucleotides used as primers for specific amplification are described in the center of the molecule (Gibbs et al. (1989) Nucleic Acids Res. 17: 2437-244).
8), or at the most 3'end of one primer where a mismatch may prevent or reduce polymerase extension under appropriate conditions (Prossner (1993) T
ibtech 11: 238) may result in the mutation of interest (so that amplification depends on differential hybridization). Furthermore, it is desirable to introduce a new restriction site into the region of mutation to perform cleavage-based detection (Gasparini).
Et al. (1992) Mol. Cell. Probes 6: 1). In a particular embodiment, amplification is also expected to be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci.
． USA 88: 189). In such cases, the ligation can detect the presence of a known mutation at a particular site by searching for the presence or absence of amplification, if there is a perfect match at the 3'end of the 5'sequence. Occurs only in.

The methods described herein can be carried out, for example, by utilizing a prepackaged diagnostic kit that includes at least one probe nucleic acid or antibody reagent described herein. Is, for example, in a clinical setting for diagnosing a patient with signs or family history of a disease or disorder involving the gene of the invention,
It can be conveniently used. Any cell type or tissue in which this gene is expressed can be utilized in the prognosis assay described herein.

3. Monitoring effects during clinical trials Expression or activity of the nucleic acid molecules or proteins of the invention (eg, regulation of cell signal transduction, regulation of gene transcription in cells involved in development or differentiation, regulation of cell proliferation). Of the effects of drugs (eg, drugs, compounds) on A.) can be applied not only in basic drug screening but also in clinical trials.
For example, the effectiveness of an agent to increase gene expression, protein levels, or protein activity, as determined by the screening assays described herein, may decrease gene expression, protein levels, or protein activity. It may be monitored in clinical trials of the subject indicated. Alternatively, the effectiveness of an agent for reducing gene expression, protein levels, or protein activity, as determined by screening assays, may be assessed in clinical trials in subjects exhibiting increased gene expression, protein levels, or protein activity. Can be monitored. In such a clinical test, the expression or activity of the identified gene, and preferably the expression or activity of other genes associated with, for example, a proliferative disorder,
It can be used as a "readout" or marker for the phenotype of a particular cell.

For example, but not intended to be limiting, treated with an agent (eg, compound, drug, or small molecule) that modulates protein activity (eg, as identified in the screening assays described herein). By doing so, one can identify the genes that are regulated in the cell. Thus, for example, in clinical trials, cells may be isolated and RNA may be prepared to study the effects of agents on proliferative disorders, disorders of development or differentiation, or hematopoietic disorders, and to identify identified genes and proliferation. Disorders, developmental or differentiation disorders, or levels of expression of other genes involved in hematopoietic disorders can be analyzed, respectively. The level of gene expression (ie, gene expression pattern) was assessed by Northern blot analysis or RT-PCR as described herein.
Or by measuring the amount of protein produced, by one of the methods described herein, or by measuring the activity level of the identified gene or other gene. obtain. Thus, the gene expression pattern can act as a marker that is an indicator of the physiological response of cells to drugs. Thus, this response state can be measured prior to treatment of the individual with the drug and at various times during treatment.

Disorders associated with programmed cell death are of particular relevance, as discussed in detail herein below.

In one embodiment, the invention provides agents (eg, agonists, antagonists, peptidomimetics, proteins, polypeptides, nucleic acids, small molecules identified by the screening assays described herein). , Or other drug candidates) for monitoring the effectiveness of treatment of the subject. The method includes the steps of: (i) obtaining a pre-dose sample from the subject prior to administration of the agent; (ii) a specified protein, mRNA, or mRNA of the invention in the pre-dose sample. Detecting the level of expression of genomic DNA; (iii) obtaining one or more post-administration samples from the subject; (iv) the level of protein, mRNA, or genomic DNA expression or activity in the post-administration sample. (V) determining the level of expression or activity of protein, mRNA, or genomic DNA in the pre-dose sample, the protein, mRNA in the post-dose sample (s),
Or comparing with genomic DNA; and (vi) altering accordingly administration of the agent to the subject. For example, it may be desirable to increase administration of the drug to increase expression or activity of the protein or nucleic acid molecule at a level higher than that detected (ie, to increase efficacy of the drug).
Alternatively, it may be desirable to reduce administration of the drug in order to reduce its effectiveness. According to such embodiments, protein or nucleic acid expression or activity can be used as an indicator of the effectiveness of the factor, even in the absence of an observable phenotypic response.

VIII. Screening Assays The present invention binds to the nucleic acid molecules, polypeptides or proteins described herein, or, for example, the expression or activity of the nucleic acid molecules, polypeptides or proteins of the invention. Methods for identifying modulators, ie, candidate or test compounds or agents (eg, antisense, polypeptides, peptidomimetics, small molecules or other drugs), that have a stimulatory or inhibitory effect on (as used herein “ Also referred to as "screening assay").

As an example, apoptosis-specific assays can be used to identify modulators of any target nucleic acid or protein of the invention. This protein and / or nucleic acid is associated with apoptosis. Thus, agents that regulate the level or activity of any of these nucleic acids or proteins can be identified by apoptosis-specific assays. For example, high throughput screens exist to identify apoptotic cells through the use of chromatin or cytoplasmic specific dyes. Thus, features of apoptosis, cytoplasmic compaction and chromosomal breaks can be used as markers to identify modulators of any of the genes involved in programmed cell death described herein. Other assays include activation of specific endogenous proteases, loss of mitochondrial function, cytoskeletal disruption, cell contraction, membrane hemorrhage, and D
Examples include, but are not limited to, nuclear compaction resulting from the degradation of NA.

In one embodiment, the invention screens for candidates or test compounds that bind to or modulate the activity of the proteins or polypeptides described herein or biologically active portions thereof. To provide an assay for. The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art. This method includes: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods that require deconvolution; "one bead, one compound" library methods; and affinity chromatography selections. Synthetic library methods using The biological library approach is limited to polypeptide libraries, but the other four approaches are applicable to small molecule libraries of polypeptides, non-peptide oligomers or compounds (Lam, K.
． S. (1997) Anticancer Drug Des. 12: 145)
.

Examples of methods for the synthesis of molecular libraries can be found in the art. For example, D
e Witt et al. (1993) Proc. Natl. Acad. Sci. U. S.
A. 90: 6909; Erb et al. (1994) Proc. Natl. Acad. S
ci. U. S. A. 91: 11422; Zuckermann et al. (1994) J.
． Med. Chem. 37: 2678; Cho et al. (1993) Science.
261: 1303; Carell et al. (1994) Angew. Chem. Int
． Ed. Engl. 33: 2059; Carell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33: 2601; and Gallop et al. (
1994) J. Med. Chem. At 37: 1233.

Compound libraries are available in solution (eg, Houghten (1992)).
Biotechniques 13: 412-421), or beads (Lam).
(1991) Nature 354: 82-84), Chip (Fodor (19
93) Nature 364: 555-556), bacteria (Landner US Pat. No. 5,223,409), spores (Ladner USP'409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. U.
S. A. 89: 1865-1869) or on phages (Scott and S
mith (1990) Science 249: 386-390); (Devl
in (1990) Science 249: 404-406); (Cwirla
(1990) Proc. Natl. Acad. Sci. 97: 6378-63
82); (Felici (1991) J. Mol. Biol. 222: 301-).
310); (Landner supra).

In one embodiment, the assay is a cell-based assay. In this assay, cells expressing the encoded polypeptide (eg, cell surface proteins such as receptors) are contacted with the test compound and the ability of the test compound to bind to the polypeptide is determined. For example, the cells can be of mammalian origin (keratinocytes). Determining the ability of a test compound to bind to a polypeptide can be accomplished, for example, by coupling the test compound with a radioisotope or an enzymatic label. As a result, the binding of the polypeptide of the test compound, by detecting with either ^{1 25 I, 35 S, 14} C or ^{3 H} in labeled either directly or indirectly ^ones, The radioisotope can be determined and detected by direct counting of radioactive emissions or by scintillation counting. Alternatively, the test compound can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of the appropriate substrate to product.

It is also within the scope of this invention to determine the ability of a test compound to interact with a polypeptide without labeling any of this interaction. For example, a micromedical instrument can be used to detect an interaction between a test compound and a polypeptide without labeling either the test compound or the polypeptide. McConne
11 et al. (1992) Science 257: 1906-1912. As used herein, "micromedical instrument" (eg, Cytosens).
or ^TM ) is an analytical instrument that measures the rate at which cells acidify their environment using a photo-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between the ligand and the polypeptide.

[0178] In one embodiment, the assay is performed by treating cells (eg, receptors) expressing the encoded proteins described herein at the cell surface with a polypeptide ligand or a biologically active portion thereof. Contacting with a test compound to form an assay mixture, contacting the assay compound with a test compound, and determining the ability of the test compound to interact with the polypeptide, wherein the test compound is The ability of the test compound to preferentially bind to this polypeptide when the step of determining its ability to interact with the polypeptide is compared to the ability of the ligand or biologically active portion thereof to bind to this polypeptide. Including the step of determining

[0179] In another embodiment, the assay is a cell-based assay, which comprises:
Determining contacting a cell expressing a particular target molecule described herein with a test compound, as well as the ability of the test compound to modulate or alter (eg, stimulate or inhibit) the activity of the target molecule. The step of performing is included. This test compound
Determining the ability to modulate the activity of a target molecule can be accomplished, for example, by determining the ability of a known ligand to bind or interact with the target molecule. Determining the ability of a known ligand to bind or interact with a target molecule can be accomplished by one of the methods described above for determining direct binding. In one embodiment, determining the ability of a known ligand to bind or interact with a target molecule can be accomplished by determining the activity of that target molecule. For example, the activity of the target molecule can be detected by detecting the induction of cellular second messengers of the target (eg, intracellular Ca ²⁺ , diacylglycerol, IP ₃ etc.), the catalytic / enzymatic activity of the target to an appropriate substrate. Detecting, inducing a reporter gene, including a target-responsive regulatory element operably linked to a nucleic acid encoding a detectable marker (eg, luciferase), or a cellular response (eg, development, Differentiation, or proliferation rate) can be determined.

In yet another embodiment, the assay of the present invention is a cell-free assay, wherein the protein of the present invention or biologically active portion thereof is contacted with a test compound and the test compound thereof. Its ability to bind to the protein or its biologically active portion is determined. The binding of this test compound to this protein is
As mentioned above, it can be determined either directly or indirectly. In one embodiment, the assay comprises contacting a protein or biologically active portion thereof with a known compound that binds to the protein to form an assay mixture, contacting the assay mixture with a test compound. And determining the ability of the test compound to interact with the protein. The step of determining the ability of the test compound to interact with the protein determines the ability of the test compound to preferentially bind to the protein or its biologically active portion as compared to a known compound. The step of performing is included.

In another embodiment, the assay is a cell-free assay, wherein a protein of the invention or biologically active portion thereof is contacted with a test compound, and the test compound is the protein or its protein. The ability to modulate or alter (eg, stimulate or inhibit) the activity of the biologically active moiety is determined. Determining the ability of this test compound to modulate the activity of this protein can be accomplished, for example, by
The ability to bind a known target molecule can be achieved by determining by one of the above methods for determining direct binding. Determining the ability of this protein to bind a target molecule is also a real-time Biomolecular In
It can be achieved using techniques such as teraction analysis (BIA). Sjolander and Urbanicky (1991) Anal
． Chem. 63: 2338-2345 and Szabo et al. (1995) Cur.
r. Opin. Struct. Biol. 5: 699-705. As used herein, "BIA" is any interactants (e.g., BIAcore T ^M), without labeling is a technology for studying biospecific interactions in real time. Optical Phenomenon Surface Plasmon Resonance (SPR)
Changes in a) can be used as indicators of real-time reactions between biological molecules.

In an alternative embodiment, determining the ability of the test compound to modulate the activity of a protein of the invention can be accomplished by determining the ability of the protein to further modulate a target molecule. For example, the catalytic / enzymatic activity of the target molecule on the appropriate substrate can be determined as described above.

In yet another embodiment, the cell-free assay involves contacting a protein of the invention or a biologically active portion thereof with a known compound that binds to the protein to form an assay mixture. Contacting the assay mixture with a test compound and determining the ability of the test compound to interact with the protein, wherein determining the ability of the test compound to interact with the protein comprises , Determining the ability of this protein to bind preferentially to a target molecule or to modulate the activity of that target molecule.

The cell-free assay of the present invention is acceptable for use with both soluble or membrane-bound forms of isolated proteins. For cell-free assays in which a membrane-bound form of the isolated protein is used, it is desirable to utilize a solubilizer so that the membrane-bound form of the isolated protein is maintained in solution. obtain. Examples of such solubilizers include nonionic surfactants such as n-octyl glucoside, n-dodecyl glucoside, n-dodecyl maltoside, octanoyl-.
N-methylglucamide, decanoyl-N-methylglucamide, Triton (registered trademark) X-100, Triton (registered trademark) X-114, Thesit (registered trademark), isotridecyl poly (ethylene glycol ether) _n (Isotr)
decypoly (ethylenglycol ether) _n ), 3
-[(3-Colamidopropyl) dimethylammonio] -1-propanesulfonate (3-[(3-cholamidopropyl) dimethylmethyl
neo] -1-propane sulphonate) (CHAPS), 3- [
(3-Colamidopropyl) dimethylammonio] -2-hydroxy-1-propanesulfonate (3-[(3-cholamidopropyl) dimension
ylamminio] -2-hydroxy-1-propane sulfo
nate) (CHAPSO), or N-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate.

In more than one embodiment of the above assay methods of the invention, either the protein or its target molecule is immobilized to allow for the formation of complexed forms from uncomplexed forms of one or both of the proteins. It may be desirable to facilitate separation and apply for automation of the assay. Binding of the test compound to the protein, or the interaction of the protein with the target molecule in the presence and absence of the candidate compound, can be accomplished in any vessel suitable for containing these reactants. . Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the proteins to bind to the matrix. For example, a glutathione-S-transferase fusion protein can be prepared using glutathione sepharose beads (Sigma Chemical, St. L.).
ouis, MO) or glutathione derivatized microtiter plates, which are then combined with the test compound or with the test compound and either non-adsorbed target protein or protein of the invention, and The conditions under which this mixture contributes to complex formation (eg salt and p
Incubated under physiological conditions for H). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components and, in the case of beads, to immobilize the matrix, e.g. It is decided either by the target. Alternatively, the complex can be dissociated from the matrix and the level of binding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the protein of the invention or its target molecule can be immobilized utilizing the conjugation of biotin and streptavidin. Biotinylated proteins or target molecules of the invention can be prepared from biotin NHS (N-hydroxysuccinimide) using techniques well known in the art (eg, biotinylation kit, Pierce Che.
micals, Rockford, IL), and streptavidin-coated 96-well plates (Pierce Chemical). Alternatively, an antibody that is reactive with the protein or target molecule of the invention but does not interfere with the binding of the protein to its target molecule can be derivatized to the wells of the plate and the unbound target or protein is , Can be trapped in the wells by conjugation of antibodies. Methods for detecting such complexes include, in addition to those described above for GST-immobilized complexes, immunodetection of the complex using an antibody reactive with the protein or target molecule, as well as the protein or target molecule. Enzyme-linked assays that rely on the detection of enzyme activity for.

In another embodiment, modulators of expression of the nucleic acid molecules of the invention are identified in a manner where a cell is contacted with a candidate compound and the expression of the appropriate mRNA or protein in the cell is determined. Appropriate mRN in the presence of candidate compound
The level of A or protein expression is compared to the level of mRNA or protein expression in the absence of the candidate compound. The candidate compound can then be identified as a modulator of expression based on this comparison. For example, if the expression of mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in the absence thereof, the candidate compound is a stimulator or enhancer of mRNA or protein expression. Identified as. Alternatively, if the mRNA or protein expression is less (statistically significantly less) in the presence of the candidate compound than in the absence of the candidate compound, then the candidate compound is identified as an inhibitor of mRNA or protein expression. . The level of expression of mRNA or protein in a cell can be determined by the methods described herein for detecting mRNA or protein.

In yet another aspect of the invention, the proteins of the invention may be used as a “bait protein” in a two-hybrid or three-hybrid assay (eg US Pat. No. 5,283,317; Zerv
Os et al., (1993) Cell 72: 223-232; Madura et al., (1
993) J Biol Chem 268: 12046-12054; Bart.
el et al., (1993) Biotechniques 14: 920-924; I.
wabuchi et al. (1993) Onconge 8: 1693-1696; and Brent WO94 / 10300), other proteins that bind to or interact with the proteins of the invention and regulate their activity (entrapped). Protein). Such captured binding proteins also
For example, it appears to be involved in the propagation of signals by the proteins of the invention as elements upstream or downstream of protein-mediated signal transduction pathways. Alternatively,
Such trapped proteins appear to be cell surface molecules associated with non-protein expressing cells, where such trapped proteins are involved in signal transduction.

The present invention further relates to novel agents identified by the above screening assay. Therefore, it is within the scope of the present invention to further use the reagents identified as described herein in a suitable animal model. For example, an agent identified as described herein (eg, a regulatory reagent, an antisense nucleic acid, a specific antibody, or a protein binding partner) can have efficacy, toxicity, or toxicity of treatment with such agents. It can be used in animal models to determine side effects. Alternatively, agents identified as described herein can be used in animal models to determine the mechanism of action of such agents. Furthermore, the present invention provides for the novel agents identified by the above screening assays,
It relates to a use for treatment as described herein.

IX. Methods of Treatment The present invention provides, or has a risk (or suspect) of a disorder associated with aberrant expression or activity of the nucleic acids of the invention or aberrant expression or activity of related proteins. Both prophylactic and therapeutic methods of treatment of certain subjects are provided.
The method of treatment involves modulating the level or activity of a nucleic acid or polypeptide in a subject with a disorder that can be treated by such modulation. Thus, modulation can result in upregulation or downregulation of nucleic acid or protein expression or upregulation or downregulation of activity. Disorders associated with programmed cell death are particularly relevant, as discussed in detail herein below.

Expression of the nucleic acids of the invention was found in the following tissues, as shown in Figure 8: prostate, brain, heart, kidney, skeletal muscle, spleen, lung, smooth muscle, pancreas, and liver. Thus, disorders to which the methods disclosed herein are particularly relevant include disorders involving these tissues.

Spleen-related disorders include nonspecific, acute pancreatitis, splenomegaly, including congestive splenomegaly, and purulent infarction; neoplasms, congenital malformations, and rupture, including Not limited to. Disorders associated with splenomegaly include the following: infections (eg, nonspecific pancreatitis, infectious mononucleosis, tuberculosis, typhoid fever, brucellosis, cytomegalovirus, syphilis, malaria, histoplasmosis, Toxoplasmosis, calazar, trypanosomiasis, schistosomiasis, leishmaniasis,
And echinococcosis); congestive conditions associated with partial hypertension (eg,
Cirrhosis, portal or splenic vein thrombosis, and heart failure; lymphohaematopoietic disorders (eg, Hodgkin's disease, non-Hodgkin's lymph / leukemia, multiple myeloma, myeloproliferative disorders, hemolytic anemia, and idiopathic thrombocytopenia) Purpura); immunological inflammatory conditions (eg, rheumatoid arthritis, and systemic lupus erythematosus); storage disorders (eg, Gaucher disease, Niemann-Pick disease, and mucopolysaccharidosis); and other conditions (eg,
Amyloidosis, primary neoplasms and cysts, and secondary neoplasms).

Disorders associated with the lung include, but are not limited to: congenital malformations; atelectasis; vascular origin disorders such as pulmonary artery congestion and edema (hemodynamic pulmonary edema and (Including edema caused by microvascular injury), adult respiratory distress syndrome (diffuse alveolar injury), pulmonary embolism, hemorrhage and infarction, and pulmonary hypertension and pulmonary arteriosclerosis); chronic obstructive pulmonary disease ( For example, emphysema, chronic bronchitis, bronchial asthma, and bronchiectasis); diffuse interstitial (wet, restrictive) disease (
For example, pneumoconiosis, sarcoidosis, idiopathic pulmonary fibrosis, exfoliative pneumonia, hypersensitivity pneumonia, pulmonary eosinophilia (pulmonary infiltration with eosinophilia), Bronchiolit
is obliterans organizing pneumonia, diffuse pulmonary bleeding syndrome (including Goodpasture's syndrome, idiopathic pulmonary hemorrhage, and other bleeding syndromes, lung involvement in collagen vascular disorders, and alveolar proteinosis); Complications (eg, drug-induced lung disease, radiation-induced lung disease, and lung transplantation); tumors (eg, bronchoprogenitor cancer (including neoplasia-associated syndrome), bronchoalveolar adenocarcinoma, neuroendocrine tumors (eg, bronchial carcinoid) ), Miscellaneous tumors, and metastatic tumors); pleural pathology (inflammatory pleural effusion, non-inflammatory pleural effusion, pneumothorax, and pleural tumor (
Includes solitary fibrous tumors (pleural fibroma) and malignant melanoma).

Liver-related disorders include, but are not limited to:
Liver injury; jaundice and cholestasis (eg bilirubin and bile formation); liver failure and cirrhosis (eg cirrhosis, portal hypertension (including ascites, portal vein anastomosis, and splenomegaly)); Infectious disorders (eg, viral hepatitis (including infection with hepatitis AE and other hepatitis viruses)), clinicopathologic syndromes (eg, carrier status, subclinical infections, acute hepatitis virus, chronic hepatitis virus, and fulminant disease) Hepatitis)); autoimmune hepatitis; drug-induced and toxin-induced liver diseases (eg,
Alcoholic liver disease); inborn errors of metabolism and childhood liver disease (eg, hemochromatosis, Wilson disease, α ₁ -antitrypsin deficiency, and neonatal hepatitis); intrahepatic bile duct disease (eg, secondary biliary cirrhosis, Primary biliary cirrhosis, primary sclerosing cholangitis, and bile duct malformation; circulatory disease (eg, impaired blood flow to the liver (including hepatic artery defects and portal vein obstruction and thrombosis), liver-mediated) Impaired blood flow (including passive congestion and centrilobular necrosis and purpura of the liver), hepatic vein outflow obstruction (including hepatic venous thrombosis (Budd-Chiari syndrome) and vein occlusion disease); liver disease associated with pregnancy (For example, preeclampsia and icterus, acute fatty liver of pregnancy, and intrahepatic cholestasis of pregnancy); liver complications of organ or bone marrow transplantation (
For example, drug toxicity after bone marrow transplantation, graft versus host disease and liver rejection, and non-immune disease damage to liver allografts; tumors and tumor conditions (eg, nodular hyperplasia, adenomas, and malignant tumors (of the liver). (Including the primary cancer of metastatic cancer) and liver fibrosis).

Brain-related disorders include, but are not limited to, neuron-related disorders and glial-related disorders such as astrocytes, oligodendrocytes, ependymal cells, and small. Glial cells); cerebral edema resulting from intracranial pressure and herniation and hydrocephalus; malformations and developmental disorders (eg neural tube defects, forebrain malformations, posterior fossa malformations, and syringomyelia and hydromyelopathy); periodic brain Injury; cerebrovascular disease (eg, hypoxia, ischemia, and infarction-related disorders (hypotension, hypoperfusion, and hypoflow conditions (global and regional cerebral ischemia)), local blood supply Intracranial hemorrhage (including intracerebral (intraparenchymal) hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysm, and aortic malformation), hypertensive cerebrovascular disease (fossil infarction, slit hemorrhage) , And hypertensive arteriosclerosis)); infections (eg, acute meningitis (including acute purulent (bacterial) meningitis and acute aseptic (viral) meningitis), acute purulent infection (brain tumor, scleroderma) Submembrane abscess and epidural abscess), chronic bacterial meningoencephalitis (including tuberculosis and mycobacteriosis), neurosyphilis, and neuroborreliosis (Lyme disease), viral meningoencephalitis (arthropod) Bone (Arbo) virus encephalitis,
Herpes simplex virus type 1, Herpes simplex virus type 2, Virtual la-zoster virus (Herpes zo
ster), cytomegalovirus, poliomyelitis, rabies, and human immunodeficiency virus 1 (including HIV-1 meningoencephalitis (subacute encephalitis)), vacuolar myelopathy, AIDS.
Associated myelopathy, peripheral neuropathy, and childhood AIDS, progressive multifocal leukoencephalopathy,
Subacute sclerosing panencephalitis, fungal meningoencephalitis, other infectious diseases of nervous system); transferable spongiform encephalopathy (prion disease); demyelinating disease (multiple sclerosis, multiple sclerosis mutants, acute dissemination) Encephalomyelitis and acute necrotizing hemorrhagic encephalomyelitis, and other demyelinating diseases; degenerative diseases (eg, degenerative diseases affecting the cerebral cortex (including Alzheimer's disease and Pick's disease), brain stem Degenerative diseases of the ganglia and brainstem (tremor paralysis,
Idiopathic Parkinson's disease (including tremor palsy), progressive supranuclear palsy, cortical basal degeneration, multiple atrophy (striatal substantia nigra, Shy-Drager syndrome, and olive pontine cerebellar atrophy, and Huntington's disease) )); Spinocerebellar degeneration (including spinocerebellar ataxia (including Friedreich's ataxia, and ataxia-telangiectasia), degenerative diseases that affect motor nerves (muscular atrophy lateral sclerosis (motor neuron disease)) , Spinal cord atrophy (Kennedy's syndrome), and spinal muscular atrophy)); , And Canavan's disease),
Cerebral myocarditis (including Leigh's disease and other cerebral myocarditis); addictive and acquired metabolic disorders (including vitamin deficiency (eg, thiamine (vitamin B1) deficiency and vitamin B ₁₂ deficiency)), metabolic disturbing nerves Sequelae (including hypoglycemia, hyperglycemia, and hepatic encephalopathy), toxic disorders (carbon monoxide, methanol, ethanol, and radiation, including a combination of methotrexate and radiation-induced injury); tumors (eg, nerves) Glioma (including astrocytoma (including fibrillar (diffuse) astrocytoma and glioblastoma pleomorphism)), pilocytic astrocytoma, pleomorphic xeroastrocytoma, and Brain stem glioma, oligodendroglioma, and ependymoma and associated paraventricular mass injury, neuronal tumors, anaplastic neoplasms (including medulloblastomas), other parenchymal tumors (primitive brain lymphoma), Grim thin Tumors, and pineal parenchymal tumors, meningiomas, metastatic tumors, paraneoplastic syndromes, peripheral nerve sheath tumors (schwannomas, neurofibromas, and malignant peripheral nerve sheath tumors (malignant nerve sheath tumors) And neurocutaneous syndrome (nevus) (including neurofibromatosis (including neurofibromatosis type 1 (NF1) and TYPE2 neurofibromatosis (NF2))), tuberous sclerosis, and Von Hippel -Lindau disease)
.

Diseases involving the heart include, but are not limited to, heart failure, including but not limited to cardiac hypertrophy, left heart failure, and right heart failure; ischemic heart disease (angina pectoris). , Myocardial infarction, chronic ischemic heart disease and sudden cardiac death; hypertensive heart disease (including but not limited to systemic (left) hypertensive heart disease and pulmonary (right) hypertensive heart disease) Valvular heart disease (valve degeneration caused by calcification (eg, calcification aortic stenosis, congenital bicuspid aortic valve calcification, and mitral annulus calcification, and mitral valve myxoid degeneration ( Mitral valve prolapse)
, Rheumatic fever and rheumatic heart disease, infectious endocarditis, and uninfected warts (eg, non-bacterial thrombotic endocarditis and systemic lupus erythematosus endocarditis (
Libman-Sachs disease), carcinoid heart disease, and heart valve complications); but myocardial disease (including dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, and myocarditis) (Including but not limited to); pericardial disease (including pericardial effusion and pericardial hematoma and pericarditis (including acute pericarditis and curative pericarditis), and rheumatoid heart disease) ); Neoplastic heart disease, including, but not limited to, primary heart tumors (eg, myxoma, lipoma, papillary elastic fibroma, rhabdomyosarcoma, and sarcoma), and cardiac effects of non-cardiac neoplasms ); Congenital heart disease (left-right shunt-late-onset cyanosis (eg, atrial septal defect, ventricular septal defect, arterial duct and atrioventricular septal defect), right-left shunt-early-onset cyanosis (
For example, Tetralogy of Fallot, Aortic Transposition, Common Artery, Tricuspid Regurgitation, and Common Pulmonary Venous Reflux Disease, Congenital Congenital Abnormalities (eg, Aortic Coarctation, Pulmonary Valve Stenosis, and Pulmonary Artery) Valve closure, and aortic stenosis and aortic valve closure), and disorders related to heart transplantation).

Disorders related to the kidney include, but are not limited to: congenital abnormalities (including but not limited to: renal cyst disease (cystic nephropathy, autosomal dominant (adult) polyps). Cystic kidney disease, including but not limited to autosomal recessive (pediatric) polycystic kidney disease, and renal medullary follicular disease (including but not limited to medullary cavernous kidneys), and nephron-neurotoxic Nephronophthisis-uremic medullar
y cystic disease complex, acquired (dialysis-related) cystic disease (eg, simple follicles); glomerular disease (pathology of glomerular damage, including but not limited to in situ immune complex deposition) anti-GBM nephritis, Ayman Nephritis, and antibodies to planted antigens, circulating immune complex nephritis, antibodies to glomerular cells, cell-mediated immunity in glomerulonephritis, activation of alternative complement pathways,
Epithelial cell damage, and pathology related to mediators of glomerular damage (including cellular mediators and soluble mediators), acute glomerulonephritis (eg, acute proliferative (poststreptococcal, postinfection) glomerulonephritis (streptococcus) Postinfectious glomerulonephritis and non-streptococcal acute glomerulonephritis, rapidly progressive (crescent formation) glomerulonephritis, nephrotic syndrome, membranous glomerulonephritis (membranous nephropathy), minimal change disease (lipoid nephrosis), Focal segmental glomerulosclerosis, membranous proliferative glomerulonephritis, IgA nephropathy (Buerger's disease), focal proliferative necrotizing glomerulonephritis (focal glomerulonephritis), hereditary nephritis (Alport syndrome and thin film) Diseases (including but not limited to benign familial hematuria), chronic glomerulonephritis, glomerular damage associated with systemic diseases (systemic lupus erythematosus, henopho-
Schoenlein purpura, bacterial endocarditis, diabetic glomerulosclerosis, amyloidosis, fibrotic and immunotactoid glomerulonephritis, and other systemic disorders); in the tubules and interstitium Affected diseases, including but not limited to acute tubular necrosis and tubular interstitial nephritis (including but not limited to pyelonephritis and urinary tract infections, acute pyelonephritis, chronic pyelonephritis and reflux nephropathy), and drugs and toxins Renal tubular interstitial nephritis (drug-induced interstitial nephritis, analgesic nephropathy, nephropathy associated with nonsteroidal anti-inflammatory drugs, and other tubular interstitial diseases (urate nephropathy, high calcium Illness and nephrocalcinosis, and including but not limited to multiple myeloma); vascular disorders (benign nephrosclerosis, malignant hypertension and And accelerated renal sclerosis, renal artery stenosis, and thrombotic microangiopathy (classical (pediatric) hemolytic uremic syndrome, adult hemolytic uremic syndrome / thrombotic thrombocytopenic purpura, idiopathic HUS / TTP , And other vascular diseases, including but not limited to atherosclerotic ischemic kidney disease, atherosclerotic kidney disease, sickle cell disease nephropathy, diffuse cortical necrosis, and renal infarction); Obstruction (obstructive uropathy); urolithiasis (calculi, stone); and tumors of the kidney (benign tumors (eg, renal papillary adenoma, renal fibroma or hamartoma (renal medulla)).
stromed) stromal cell tumor), angiomyolipoma, and oncocytoma)
, And malignant tumors (including but not limited to renal cell carcinoma (including adrenal tumor, adenocarcinoma of the kidney) (including urothelial carcinoma of the pelvis)).

Disorders of the testes and epididymis include, but are not limited to: congenital abnormalities (eg, latent testicular testicularism), retrograde changes (eg, atrophy), inflammation (eg, nonspecific). Epididymitis and orchitis), granulomatous (autoimmune) orchitis, and specific inflammation (including but not limited to gonorrhea, mumps, tuberculosis, and syphilis), vasculopathy (twisting) , Including testicular tubular adenoma (
(Including but not limited to germ cell tumors) including but not limited to seminoma, spermatocyte seminoma, embryonal carcinoma, yolk sac tumor cystic carcinoma, teratoma, and mixed tumors) Tumors, including but not limited to Leydig cell tumors and Sertoli cell tumors (androgen producing cell tumors)
, And testicular lymphoma, and other lesions of the sheath.

[0199]   Skeletal muscle disorders include tumors such as rhabdomyosarcoma.

Disorders related to the pancreas include the following: disorders of the exocrine pancreas (eg,
Congenital abnormalities (including but not limited to ectopic pancreas); pancreatitis (including but not limited to acute pancreatitis); cysts (including pseudocysts,
Tumors (including but not limited to sac tumors and cancers of the pancreas); and disorders of the endocrine pancreas (eg, diabetes); islet cell tumors (
Insulinoma, gastrinoma, and other rare islet cell tumors).

[0201]   Preferred disorders include disorders of the central nervous system and especially the brain.

With respect to both prophylactic and therapeutic methods of treatment, such treatments are tailored based on knowledge gained from the field of pharmacogenomics.
Can be obtained or modified. “Pharmacogenomics” as used herein refers to gene sequencing, statistical genetics, for drugs in clinical development and marketing.
And the application of genomics techniques such as gene expression analysis. More specifically, this term refers to how a patient's genes determine his or her response to a drug (eg, a patient's "drug response phenotype" or "drug response genotype"). Refers to research. Accordingly, another aspect of the invention provides a method for tailoring an individual prophylactic or therapeutic treatment with a molecule or modulator of the invention according to its individual drug response genotype. Pharmacogenomics allows clinicians or physicians to target prophylactic or therapeutic treatments to patients who will most benefit from this treatment, and avoid treatment of patients who experience side effects associated with toxic drugs. obtain.

(1. Prevention Method) In one aspect, the present invention provides a method of administering to a subject an agent that regulates expression or at least one activity of the gene or protein of the present invention. Methods are provided for preventing diseases or conditions associated with aberrant expression or activity of a gene or protein of the invention. A subject at risk of a disease caused by or resulting from aberrant gene expression or protein activity may be identified by, for example, any of the diagnostic or prognostic assays described herein or a combination thereof. .. Administration of the prophylactic agent can occur before the appearance of abnormally characteristic symptoms, thereby preventing or delaying the progression of the disease or disorder. Depending on the type of abnormality, for example, an agonist or antagonist agent can be used to treat the subject. The appropriate agent can be determined based on the screening assays described herein.

2. Therapeutic Method Another aspect of the present invention relates to a method of regulating the expression or activity of the gene or protein of the present invention for therapeutic purposes. The method of regulation of the invention involves contacting a cell with an agent that regulates one or more activities of a particular protein associated with that cell. Agents that modulate protein activity include agents described herein, eg, nucleic acids or proteins, naturally occurring target molecules of the proteins described herein, polypeptides, peptidomimetics, or other It can be a small molecule. In one embodiment, the agent stimulates one or more protein activities. Examples of such stimulants include active proteins and nucleic acid molecules that encode proteins introduced into cells. In another embodiment, the agent inhibits one or more protein activities. Examples of such inhibitory agents include antisense nucleic acid molecules and anti-protein antibodies. These methods of modulation can be performed in vitro (eg, by culturing cells with the drug) or in vivo (eg, by administering the drug to a subject). Thus, the invention provides a method of treating an individual suffering from a disease or disorder characterized by aberrant expression or activity of a protein or nucleic acid molecule of the invention. 1
In one embodiment, the method modulates expression (eg, upregulation or downregulation) of an agent (eg, an agent identified by a screening assay described herein), or a gene or protein of the invention. Controlling) administering a combination of agents. In another embodiment, the method comprises administering a protein or nucleic acid molecule of the invention as a treatment to compensate for reduced or aberrant expression or activity of the protein or nucleic acid molecule.

Stimulation of protein activity is desirable in situations where the protein is abnormally downregulated and / or where increased protein activity may have a beneficial effect. Similarly, inhibition of protein activity is desirable in situations where the protein is abnormally upregulated and / or where decreasing protein activity may have a beneficial effect. One example of such a situation is one in which the subject has a disorder characterized by abnormal development or cell differentiation. In another example of such a situation, the subject has a proliferative disease (eg, cancer) or a disorder characterized by an abnormal hematopoietic response. In yet another embodiment of such circumstances, it is desired to achieve tissue regeneration in the subject (eg, where the subject has brain or spinal cord injury, it regenerates neuronal tissue in a regulated manner). Desirable).

Pharmaceutical Compositions Nucleic acid molecules, proteins, modulators of this protein, and antibodies (also referred to herein as “active compounds”) are suitable for administration to a subject (eg, human). It can be incorporated into a pharmaceutical composition. Such compositions typically include a nucleic acid molecule, protein, modulator, or antibody, and a pharmaceutically acceptable carrier.

The term “administering” is used in its broadest sense and includes any method of introducing the composition of the present invention into a subject. This includes production of the polypeptide or polynucleotide in vivo, such as by in vivo transcription or translation of the polynucleotide exogenously introduced into the subject. Thus, a polypeptide or nucleic acid produced in a subject from an exogenous composition is included in the term "administering".

As used herein, the language “pharmaceutically acceptable carrier” refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal compatible with pharmaceutical administration. Agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except to the extent of any conventional vehicle or drug that is incompatible with the active compound, such vehicles may be used in the compositions of the invention. Supplementary active compounds can also be incorporated into the compositions. A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration are parenteral (eg intravenous), intradermal, subcutaneous, oral (eg inhalation), transdermal (topical).
, Transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following ingredients: sterile diluent (eg water for injection, saline solution, fixed oils, polyethylene). Glycol, glycerin, propylene glycol or other synthetic solvents); antibacterial agents (eg benzyl alcohol or methylparaben); antioxidants (eg ascorbic acid or sodium bisulfite); chelating agents (eg ethylenediaminetetraacetic acid)
Buffers (eg, acetate, citrate, or phosphate), and agents for adjusting tonicity (eg, sodium chloride or glucose). pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Suitable carriers for intravenous administration include saline, bacteriostatic water, Cremophor EX ^™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should have easy syringability.
It should be fluid to the extent that it exists. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols such as glycerol, propylene glycol, and liquid polyethylene glycols, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by combining the one or a combination of the ingredients listed above with the required amount of the active compound (eg, ubiquitin protease protein or anti-ubiquitin protease antibody) in a suitable solvent. , Followed by sterile filtration if necessary. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is to obtain a powder of the active ingredient and any further desired ingredients from the solution which have been previously sterile filtered, vacuum drying and freeze drying. Is.

Oral compositions generally include an inert diluent or an edible carrier. They are,
They can be enclosed in gelatin capsules or compressed into tablets. For oral administration, the drug may be included in enteric form to remain in the stomach or may be further coated by known methods or mixed to be released in specific areas of the GI tract. Can be done. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and exhaled. Be swallowed or swallowed. Pharmaceutically compatible binders, and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches, etc. may contain a compound having any of the following ingredients or similar properties: binders (eg microcrystalline cellulose, gum tragacanth or gelatin); excipients (eg Starch or lactose), disintegrants (eg alginic acid), Primogel, or corn starch; lubricants (eg magnesium stearate or Sterot)
es); glidants (eg, colloidal silicon dioxide)
A sweetener (eg sucrose or saccharin); or a flavoring agent (eg peppermint, methyl salicylate, or orange flavor).

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished by nasal spray or suppositories. For transdermal administration, the active compound may be an ointment, which is generally known in the art,
Formulated in ointment, gel, or cream.

The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compound is prepared with a carrier that protects the compound against rapid elimination from the body (eg, controlled release formulations),
This includes implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
Methods for the preparation of such formulations will be apparent to those of skill in the art. These materials are also commercially available from Alza Corporation and Nova Phar.
scientifics, Inc. Commercially available from Liposomal suspensions, including liposomes targeted to infected cells, containing monoclonal antibodies against viral antigens, can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in US Pat. No. 4,522,811.

It is especially beneficial to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, a "dosage unit form" is suitable as a unit dosage for the subject to be treated,
Physically refers to individual units; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Details regarding the dosage unit forms of the present invention are given by the unique properties of the active compound, and the particular therapeutic effect achieved, as well as limitations inherent in the art of formulating such active compounds for treatment of individuals. Determined and depends directly on these.

The nucleic acid molecule of the present invention can be inserted into a vector and used as a gene therapy vector. Gene therapy vectors are, for example, intravenous injection, local administration (US Pat. No. 5,328,470) or stereotactic injection (eg Chen et al. (1994).
) PNAS 91: 3054-3057)). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, the complete gene delivery vector can be produced intact from recombinant cells (eg, a retroviral vector) and the pharmaceutical preparation can include one or more cells that produce a gene delivery system.

The pharmaceutical composition can be included in a container, package, or dispenser together with instructions for administration.

As defined herein, a therapeutically effective amount (ie, effective dosage) of a protein or polypeptide is about 0.001-30 mg / kg body weight, preferably about 0.01-25 mg. / Kg body weight, more preferably about 0.1-20 mg / kg
Body weight, and even more preferably about 1-10 mg / kg, 2-9 mg / kg, 3
-8 mg / kg, 4-7 mg / kg, or 5-6 mg / kg body weight.

Those skilled in the art will recognize that certain factors, including the severity of the disease or disorder, previous treatment, the general health and / or age of the subject, and other diseases present, (But not limited to) can affect the dosage required to effectively treat a subject. In addition, a therapeutically effective amount of the protein, polypeptide,
Alternatively, treatment of a subject with an antibody can include a single treatment or, preferably, can include a series of treatments. In a preferred example, the subject is about 0.1-20 mg.
/ Kg body weight in the range of about 1 to 10 weeks, preferably 2 to 8 weeks, more preferably about 3 to 7 weeks, and even more preferably about 4, 5 or 6 weeks. Treat with antibody, protein, or polypeptide once a week for a week. It is also understood that the effective dosage of antibody, protein, or polypeptide used for treatment may be increased or decreased over the course of a particular treatment. Changes in dosage may be caused and may be apparent from the results of clinical assays as described herein.

The present invention includes agents that regulate expression or activity. The drug can be, for example, a small molecule. For example, such small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, about 10,000 grams per mole. Organic or inorganic compounds having a molecular weight of less than (
That is, including heteroorganic compounds and organometallic compounds) about 5 per mole
An organic or inorganic compound having a molecular weight of less than 1,000 grams, an organic compound or an inorganic compound having a molecular weight of less than about 1,000 grams per mole, an organic compound or an inorganic compound having a molecular weight of less than about 500 grams per mole, And salts, esters, and pharmaceutically acceptable forms of such compounds.

It is understood that the appropriate dosage of a small molecule drug depends on many factors within the knowledge of the ordinarily skilled physician, veterinarian, or investigator. Small molecule doses will depend, for example, on the identity, size, and condition of the subject or sample being treated,
Furthermore, where applicable, it will vary depending on the route of administration by which the composition is administered, as well as the effect that one of skill in the art would like to have for the nucleic acid or polypeptide of the invention with this small molecule. An exemplary dose is a subject or sample weight of 1.
An amount of milligrams or micrograms per kilogram (eg, about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram). Approximately 50 micrograms per kilogram) of small molecules. Moreover, it is understood that the appropriate dose of the small molecule depends on the potency of the small molecule with respect to the expression or activity to be regulated. Such suitable doses can be determined using the assays described herein. When one or more of these small molecules are administered to an animal (eg, a human) to modulate the expression or activity of a peptide or nucleic acid of the invention, the physician, veterinarian, or researcher first May be prescribed a relatively low dose, followed by escalation of the dose until an appropriate response is obtained. Further, the particular dose level for any particular animal subject will depend on a variety of factors (activity of the particular compound utilized, age, weight, general health, sex, and subject's diet, time of administration, administration). Route, excretion rate, any drug combination, as well as the degree of expression or activity to be regulated).

3. Pharmacogenomics Pharmacogenomics, as identified by the screening assays described herein.
The molecules of the invention, as well as factors that have a stimulatory or inhibitory effect on protein activity (eg, gene expression), ie, modulators, impair disorders associated with aberrant protein activity (eg, proliferative or developmental disorders). It can be administered to an individual for treatment (prophylactically or therapeutically). Combined with such treatment,
Pharmacogenomics, ie, studies of the relationship between an individual's genotype and that individual's response to foreign compounds or drugs, can be considered. Differences in metabolism of therapeutics can lead to severe toxicity and therapeutic failure by altering the relationship between dose and blood concentration of the pharmacologically active drug. Accordingly, a physician or clinician will decide in deciding whether to administer a molecule of the invention or a modulator thereof, and in formulating a therapeutic regimen for dosing and / or treatment with such a molecule or modulator. Applying the knowledge gained in relevant pharmacogenomics to

Pharmacogenomics deals with clinically significant hereditary alterations in the response to drugs due to altered drug properties and aberrant effects in affected individuals. For example,
Eichelbaum (1996) Clin Exp. Pharmacol. P
hysiol, 23 (10-11): 983-985 and Linder (19
97) Clin. Chem, 43 (2): 254-266. In general, two types of pharmacogenomic states can be distinguished. Is genetic status transmitted as a factor that modifies the way drugs act on the body (altered drug action)?
, Or genetic conditions are transmitted as one factor that alters the way the body acts on drugs (altered drug metabolism). These pharmacogenomic states can occur either as rare defects or as naturally occurring polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common hereditary enzymatic disease, the main clinical complications of which are oxidative drugs (antimalarials, sulfonamides, analgesics, nitrofuran). ) Ingestion and consumption of broad beans.

[Genome-wide association (genome-wide association
One known pharmacogenomics approach to identify a gene that predicts a drug response, known as "ion," is 60,000-100,000 in the human genome, already known as a gene-related marker (eg, each has two variants). We mainly rely on a high resolution map of the human genome consisting of a "bi-allelic" gene marker map consisting of polymorphic or modified sites of Such high resolution genetic maps are maps of the respective genomes of a statistically significant number of patients participating in Phase II / III drug trials to identify markers specifically associated with observed drug response or side effects. Can be compared with. Alternatively, such high-resolution maps can be used for tens of millions of known single nucleotide polymorphisms (SNs) in the human genome.
It can be made from a combination of P). As used herein, "SNP" is
It is a common change that occurs at a single nucleotide in a stretch of DNA. For example, SNPs can occur once every 1,000 bases of DNA. SNPs may be involved in disease processes, but for the most part may not be disease related. Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into gene categories that depend on the particular pattern of SNPs in their individual genomes. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, thus taking into account common traits among genetically similar individuals.

Alternatively, a method termed the “candidate gene approach”, can be utilized to identify genes that predict drug response. According to this method, where the gene encoding the drug target is known (eg, the protein or polypeptide of the invention)
, All common modifications of that gene can be fairly easily identified in that population, and whether having one version of the gene relative to another version of the gene is associated with a particular drug response Can be determined.

As an exemplary embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. Drug metabolizing enzymes (eg, N-acetyl transferase 2 (NAT 2) as well as CYP2D, which is a cytochrome P450 enzyme)
6 and CYP2C19) found that some patients either did not get the expected drug effect after taking standard and safe doses of the drug, or exaggerated drug response and Provided an explanation as to whether it is severely toxic. These polymorphisms are expressed in two phenotypes in the population (rapid metabolizer (EM) and poor metabolizer (PM). The prevalence of PM differs between different populations. For example, encoding CYP2D6. Gene is highly polymorphic and several mutations have been identified in PM, all of which are functional CYP2D6.
Leads to the nonexistence of. Poor metabolites of CYP2D6 and CYP2C19 very often experience exaggerated drug response and side effects when receiving standard doses. When the metabolite is the active therapeutic moiety, PM does not show a therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite, morphine. The other extreme is the so-called ultrarapid metabolizer, which does not respond to standard doses. In recent years, molecules based on ultrarapid metabolites have been identified to be due to CYP2D6 gene amplification.

Alternatively, a method termed “gene expression profiling”, can be utilized to identify genes that predict drug response. For example, gene expression in an animal that is dosed with a drug (eg, a molecule or modulator of the invention) may provide an indication of whether the gene pathway associated with toxicity is turned on.

The information generated from more than one of the above pharmacogenomic approaches can be used to determine appropriate dosing and treatment regimens for prophylactic or therapeutic treatment of an individual. This knowledge, when applied to dosing or drug selection, may avoid adverse reactions or therapeutic failures, and, thus, the molecules or modulators of the invention (eg, of the exemplary screening assays described herein). The modulators identified by 1) may be used to enhance therapeutic or prophylactic efficacy when treating a subject.

Disorders that can be treated or diagnosed by the methods described herein include:
Examples include disorders related to apoptosis, but are not limited thereto. Certain disorders are associated with an increased number of viable cells that are produced and continue to survive or proliferate if apoptosis is inhibited.

As used herein, "programmed cell death" refers to genetically regulated processes involved in the normal development of multicellular organisms. This process can occur in a variety of normal situations (larval development of C. elegans nematodes, insect metamorphosis, development of mammalian embryos including nephrogenic zones in the developing kidney, and regression or atrophy (eg,
(In post-castration prostate)) occurs in cells that are destined for removal (including). Programmed cell death is the removal of growth and trophic factors in many cells, lack of necessary nutrition, hormone treatment, UV irradiation, and reactive oxygen species and phosphatase inhibitors (eg okadaic acid), calcium ionophores and It can occur following exposure to toxic and infectious agents, including many cancer chemotherapeutic agents. Wilson (1998) Biochem. Cell Biol. 7
6: 573-582 and Hetts (1998) JAMA 279: 300-3.
07, the contents of which are incorporated herein by reference. Thus, the proteins of the invention may regulate programmed cell death pathway activity by being differentially expressed during programmed cell death (eg, neuronal programmed cell death), and especially in cells expressing this protein, It provides novel diagnostic targets and therapeutic agents for disorders characterized by unregulated programmed cell death.

As used herein, a “disorder characterized by deregulated programmed cell death” is a disorder characterized by deregulation of programmed cell death (eg, upregulation or downregulation). , Refers to a disease or condition. Deregulation of programmed cell death can lead to deregulation of cell proliferation and / or cell cycle progression. Examples of disorders characterized by deregulated programmed cell death include, but are not limited to, neurodegenerative disorders (eg, Alzheimer's disease), dementias associated with Alzheimer's disease (eg, Pick's disease). , Parkinson's disease and other Lewy diffuse bodies (Lewy diffuse bo)
dy) disease, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, Jakob-Kreuzfeldt disease, or AIDS-related dementia; myelodysplasia (
For example, aplastic anemia); ischemic injury (eg myocardial infarction, stroke, or reperfusion injury); autoimmune disorder (eg systemic lupus erythematosus, or immune-mediated glomerulonephritis); or proliferative disorder (eg, Cancer (eg follicular lymphoma), p5
Cancers with 3 variants, or hormone-dependent tumors (eg breast, prostate, or ovarian cancer). The clinical manifestations of defective apoptosis are also found in stroke and rheumatoid arthritis. Wilson (1998) Biochem. Ce
ll. Biol. 76: 573-582.

Failure to remove autoimmune cells that develop during development or autoimmune cells that develop as a result of somatic mutations during the immune response can result in autoimmune disease. One of the molecules that plays a key role in the regulation of lymphocyte cell death is the cell surface receptor for Fas.

Viral infections, such as those caused by herpesviruses, poxviruses, and adenoviruses, can result in abnormal apoptosis. Cell populations are also often reduced in the event of viral infections, and perhaps the most dramatic example is the cellular loss caused by the human immunodeficiency virus (HIV). HI
Most T cells that die during V infection do not appear to be HIV infected. CD4
Stimulation of the receptor causes enhanced susceptibility of uninfected T cells to undergo apoptosis.

Many disorders can be classified based on whether the disorder is associated with abnormally high or abnormally low apoptosis. Thompson (1995) Scie
nce 267: 1456-1462. Apoptosis is associated with acute injury, myocardial infarction, stroke, and infectious disorders such as viral herpes and acquired immunodeficiency syndrome.

Primary apoptotic defects include transplant rejection. Accordingly, the present invention relates to the identification of genes useful in inhibiting transplant rejection.

Primary apoptotic defects also include autoimmune diabetes. Therefore, the present invention provides
Identification of genes associated with autoimmune diabetes, and thus acting on these targets,
It is associated with the identification of agents that regulate the expression of these genes and that treat or diagnose this disorder. Furthermore, it is suggested that all autoimmune disorders can be considered as primary defects in apoptosis (Hetts (supra)). Accordingly, the present invention relates to screening for gene expression and transcription profiles in any autoimmune disease, and for agents that affect expression or transcription of these genes.

Primary apoptotic defects also include loci autoreactivity disorders. This includes Hashimoto thyroiditis.

Primary apoptotic defects also include lymphoproliferative and autoimmune. This includes but is not limited to Canal-Smith syndrome.

Primary apoptotic defects also include cancer. For example, p53 includes apoptosis by acting as a transcription factor that activates the expression of various apoptosis-mediating genes or by upregulating apoptosis-mediating genes such as Bax.

Primary hyperapoptosis is associated with neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, spinal muscular atrophy, and amyotrophic lateral sclerosis).

Primary hyperapoptotic also can be associated with idiopathic dilated cardiomyopathy, ischemic dilated cardiomyopathy,
And heart disease, including heart valve disease. This evidence indicates apoptosis in heart failure resulting from arrhythmogenic right ventricular trait abnormalities. See Hetts, above, for all of these disorders.

Death receptors are also included in TNF receptor-1, thus TNG acts as a death ligand.

The widespread neurological disease is characterized by a gradual loss of a set of characteristics of neurons. Such disorders include: various forms of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS) retinitis pigmentosa, spinal muscular atrophy, and cerebellar degeneration. Cell loss in these diseases does not involve an inflammatory response, and apoptosis appears to be the mechanism of cell death.

In addition, several hematological disorders are associated with diminished production of blood cells. These disorders include chronic diseases, aplastic anemia, chronic neutropenia, and. Includes anemia associated with myelodysplastic syndrome. Impaired blood production (eg, myelodysplastic syndrome and aplastic anemia) is associated with increased apoptotic cell death in the bone marrow.

These disorders may result from apoptosis, defective acquisition in stromal cells or hematopoietic survival factors, or activity of genes that promote direct effects of toxins and mediators of the immune response.

Two common disorders related to cell death are myocardial infarction and stroke. In both disorders, cells within the central region of ischemia (produced in the event of acute loss of blood flow) appear to die rapidly as a result of necrosis. However, outside the central ischemic region, cells appear to die over a more delayed period and die morphologically by apoptosis.

The present invention also relates to disorders of the central nervous system (CNS). These disorders include, but are not limited to, cognitive impairment and autonomic neuropathy (eg, Alzheimer's disease, senile dementia, Huntington's disease, amyotrophic lateral sclerosis, and Parkinson's disease and Jill Doh・ La Tourette syndrome, autonomic dysfunction (
For example, hypertension and sleep disorders), and neuropsychiatric disorders, including but not limited to: schizophrenia, schizoaffective disorder, attention deficit disorder, dysthymia, major depression, mania, obsessive-compulsive disorder. Sexual disorder, psychoactive substance use disorder, anxiety, panic disorder, and bipolar affective disorder (eg, severe bipolar affective (modal) disorder (B
P-1), bipolar affective (mode) disorder with hypomania and major depression (BP-II))). Furthermore, as CNS-related disorders, for example, American Ps
ychiatric Association's Diagnostica
nd Statistical of Mental Disorders (DSM
) (These recent versions are incorporated herein by reference in their entirety).

As used herein, “differential expression” or “differentially expressed”.
Is a quantitative or qualitative difference in the temporal and / or cellular expression pattern of a gene (eg, programmed cell death described herein in normal cell (s) undergoing programmed cell death). , For example, the differentially expressed gene is at risk of developing a disorder characterized by deregulated programmed cell death, and is part of a prognostic and diagnostic marker for the assessment of subjects. Can be used as. Depending on the expression level of the gene, the progression status of the disorder can also be assessed.

X. Arrays and Microarrays The term “array” refers to a set of nucleic acid sequences that comprises at least one of SEQ ID NOs: 1-6, 8 and 10. A preferred array comprises multiple genes. This term
It refers to the entire sequence of SEQ ID NOs: 1-6, 8 and 10 but may also include additional sequences, such as sequences included as controls for a particular biological process. A "subarray" is also obtained by creating an array, but less than all of the sequences in the starting array.

In one embodiment of the invention the functional sub-array comprises nucleic acid sequences expressed in programmed cell death as disclosed in the invention.

The arrays include not only the specifically designed sequences, but also variants of these sequences as described herein. As described, variants include allelic variants, homologues from other loci of the same animal, orthologs.
og), and sufficiently similar sequences, to meet the need for sequence identity / homology as described herein.

Furthermore, this array not only contains the specific designed sequences, but also fragments thereof. As described herein, the range of this fragment will vary depending on the particular sequence involved. Therefore, the range of this fragment is, for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150,
200, 250, 300, 350, 400, 450, 500, 550, 600,
650, 700, 750, 800, 850, 900, 950, 1000, etc. However, none are fragments that should be constructed as having the same sequence as can be found in the prior art.

This array can be used to assay the expression of one or more genes in the array.

In one embodiment, the array can be used to assay gene expression in tissues to confirm the tissue specificity of the genes in the array.

In addition to these qualitative measurements, the present invention allows quantification of gene expression. Therefore, not only the tissue specificity, but the expression level of the battery of genes in the tissue is ascertained. Thus, genes can be classified based on their tissue expression itself, as well as the level of expression in this tissue. For example, this is useful in identifying gene expression relationships between or within tissues. Thus, one tissue can be disrupted and the effect on gene expression of the second tissue can be determined. In this situation, the effect of one cell type on the other in the response to the biological stimulus can be measured. Such a measurement is useful for knowing cell-cell interactions, for example, at the gene expression level. When an agent is therapeutically administered to treat one cell type, but has an unwanted effect on another cell type, the present invention provides an assay for measuring the molecular basis of the unwanted effect. And thus provide an opportunity to co-administer a co-administered drug or otherwise treat an unwanted effect. Similarly, even within a single cell type, undesired biological effects can be measured at the molecular level. Therefore,
The effect of drugs on expression other than the target gene can be confirmed and neutralized.

In another embodiment, the array can be used to monitor the time course of expression of one or more genes in the array. This is as disclosed herein in a variety of biological contexts (eg development and differentiation, tumor growth, growth of other diseases, in vitro processes (eg cell transformation and senescence), autonomic nervous system). And neural processes (eg, pain and appetite), as well as congestive functions (eg, learning and memory).

This array is also useful for confirming the effect of gene expression on the expression of other genes in the same cell or different cells. This provides for the selection of alternative molecular targets for therapeutic treatment if the ultimate or downstream target cannot be regulated.

This array is also useful for confirming the differential expression pattern of one or more genes in normal and abnormal cells. It is a battery of genes that can serve as a molecular target for diagnostic or therapeutic treatments.

In one embodiment, the array (particularly a subarray comprising one or more nucleic acid sequences associated with programmed cell death) is useful for diagnosing a disease or for predisposing to a disease associated with apoptosis. . These disorders include, but are not limited to, the disorders discussed herein. Furthermore, the arrays or subarrays produced therefrom are useful for diagnosing disorders of activation of the nervous system, or for predicting the time period for developing such disorders. Central nervous system disorders include, but are not limited to, those disorders described herein. In addition, this array and its sub-arrays are useful for diagnosing active disorders, or for predicting the time period for developing disorders, including but not limited to: secretory / synaptic vesicle release, Cell proliferation, cytoskeleton remodeling, stress / hormone response; and calcium signaling.

This array is also useful for confirming expression of one or more genes in a model system in vitro or in vivo. Various model systems have been developed to study normal and aberrant processes, including but not limited to apoptosis.

Apoptosis can be actively induced by a diverse array of triggers ranging from ionizing radiation to hypothermia of viral infections to the immune response. Majno et al. (1995) Amer. J. Pathol. 146: 3-15; Hockenb
erry et al. (1995) Bio Assays 17: 631-638; Thom.
Pson et al., Science 267: 1456-1462 (1995).

A transgenic mouse model was developed for the family of amyotrophic lateral sclerosis, Alzheimer's disease and Huntington's disease (Prin.
Ce et al. (1998) Science 282: 1079-1083). Amyotrophic lateral sclerosis is the most common development of motor neuropathy in adults.
Alzheimer's disease is the most common cause of dementia in adult survival. It is associated with damage to areas of cognition and memory and to neural circuits, including the neocortex,
These include neurons in the hippocampus, tonsils, whole basal ganglia cholinergic system, and brainstem monoaminergic nuclei. Neurological disorders associated with autosomal dominant trinucleotide repeat mutations include Huntington's disease, various spinocerebellar ataxias and dentate nucleus erythematosus pallidum Louis Atrophy. SCA-1 and SCA-3 or Machado-Jos
Eph disease is characterized by ataxia and lack of coordination. In Huntington's disease, symptoms are associated with degeneration of the striatum and a subset of cortical neurons. Apoptosis is believed to play a role in the degeneration of these cells. Various cell populations and specific cells in the cerebellum have been shown to degenerate in SCA-1, SCA-3, and in dentate nucleus red nucleus pallidum Louis Atrophy. See Prince et al., Supra, which is incorporated by reference in its entirety for the teaching of model systems associated with neurodegenerative diseases.

From a mouse model, non-obese mice were developed to study disease progression for treatment of autoimmune diabetes mellitus. Bellgrau et al. (1995) Nature
e377: 630-632. A model has also been developed in the mouse, where the mouse lacks one or more copies of the p53 gene. Studies of these mice have shown that apoptosis is associated with suppression of tumor spread in vivo. Lozano et al. (1998) Semin. Canc. Biol. 8: 337
-344. Another animal model associated with apoptosis involves the study of apoptosis associated with disruption of the target gene for the caspase gene, which creates caspase gene knockout mice. Colussi et al. (1999) J. Immun. Cel
l. Biol. 77: 58-63. Additional mouse models are associated with frostbite in mice (eg, damage involving neuronal apoptosis). Murakami et al. (19
99) Prog. Neurobiol. 57: 289-299.

Knockout mice have been made with Apafl. In these mice, deletions are found essentially in all tissues (development of this tissue includes loss of interdigital webs, palate formation, regulation of neuronal number, and lens and retina development). , All tissue development depends on cell death). Ceccon
i et al. (1998) Cell 94: 727-737.

Caspase knockout mice have been achieved for caspases 1, 2, 3, and 6. Green (1998) Cell 94: 695-698.

This array allows for the simultaneous measurement of a battery of genes involved in these processes, providing multiple candidates for in vivo confirmation and clinical trials. Since this array allows the expression of multiple genes to be determined, confirming candidate gene expression provides a powerful tool, both qualitatively and quantitatively, in both time and / or tissue-specific manners. Co-express two or more genes in a specific fashion. Thus, genes can be classified based on their expression per se and / or expression level. This ensures that even if the gene is not fully characterized for function,
Allows genes to be categorized by functional category. Therefore, if the first gene is expressed in a coordinated manner with a second gene of known function, a putative function can be selected for the first gene. Therefore, the first gene provides a new target for affecting its function for diagnostic or therapeutic purposes. The greater the number of arrays, the more likely it is that multiple known genes with the same or similar function will be expressed. In this case, the coordinated expression (in terms of function and / or structure) of one or more novel genes strongly enables the discovery of genes in the same functional class as known genes.

The arrays of the invention therefore provide an “internal control” group of genes, the function of which is known and therefore, when they are expressed in a coordinated manner, are present in the same functional group of control groups. Can be used to identify such genes.

As an alternative to relying on such internal control groups, external control groups can be added to the array. Genes in such a group have known functions. Therefore, it is clear that genes co-expressed with these genes are associated with the same function.

[0270] Therefore, this array is known not only for discovering novel genes having a specific function, but also for setting a function in a gene of unknown function, or for discovering additional functions that were previously unknown with respect to the gene. To provide a method for setting the gene of.

Therefore, as disclosed and exemplified herein, a previously characterized gene was previously known to possess a novel function (ie, retained by that gene). There was no function). Furthermore, several characterized genes can be functionally grouped into "internal control groups of genes" based on coordinated expression. In the specific embodiments disclosed and exemplified herein, genes associated with programmed cell death in the brain were selected. Therefore, it can be used to select for genes associated with other important biological processes, such as those disclosed herein. Nucleic acids from any tissue in any biological process are hybridized to the nucleic acid sequences of the array. The expression patterns of the genes in this array allow their classification into functional groups based on the particular expression pattern. Internal or external control genes (ie, genes known to be expressed in a particular tissue / biological process) provide confirmation for classifying other genes in a particular class.

Indeed, where this array is useful for identifying programmed cell death genes, other relevant normal biological models are differential programs and disorders as described herein. including.

This array is also useful for drug discovery. Candidate compounds can be used to screen cells and tissues in any of the biological contexts disclosed herein (eg, pathology, development, differentiation, etc.). Thus, expression of one or more genes in the array can be monitored by using the array to screen for RNA expression in cells or tissues exposed to the candidate compound. A compound may be selected based on its overall effect on gene expression, not necessarily based on its effect on a single gene. Thus, for example, affecting a particular first gene (s), but not the second
If a compound that does not affect the gene (s) of is desired, this array provides a way to globally monitor the effect of the compound on gene expression.

Alternatively, it may be desirable to target one or more genes (ie regulate the expression of one or more genes). This array provides a way to discover compounds that regulate a set of genes. All genes in this set can be upregulated or downregulated. Alternatively, part of this gene can be upregulated and the other downregulated by the same compound. In addition, compounds that modulate the desired gene to the desired extent can be discovered.

In the drug discovery context, functional subarrays of genes are particularly useful. Thus, using the methods discovered herein and those routinely available, groups of genes can be constructed based on their association with a particular biological function. The expression of this group of genes can be used for diagnostic purposes and to discover compounds for biological function. Thus, this subarray may provide the principle for discovering drugs that are relevant to the treatment and diagnosis of diseases, such as those described herein.

In this case, the group of genes whose expression is associated with programmed cell death is
It can be used to discover compounds that affect programmed cell death and certain disorders associated with programmed cell death. These include, but are not limited to, those disclosed herein.

Apoptosis can be induced in media or in vivo by adding pro-apoptotic ligands to the cells. Thus, in one embodiment of the invention, the arrays and subarrays described herein identify the genes that respond to pro-apoptotic ligands and, conversely, the ligands that act at genes associated with apoptosis. Is useful for Apoptosis can also be induced by reducing or eliminating apoptosis-inhibiting or survival-promoting ligands. Therefore, apoptosis is triggered by the fact that this cell lacks a signal from the cell surface survival factor receptor. As a ligand,
Examples include, but are not limited to, FasL. IL as a death-inhibiting ligand
-2, but is not limited thereto. Hetts et al. (1998) JAMA
279: 300-307, which is incorporated by reference in its entirety for teaching ligands regarding active and passive apoptotic pathways. Useful as potent molecules to induce (or release from inhibition) the apoptotic pathway, the centers of this pathway include FADD, caspases, the human CED4 homolog (also called apoptotic protease activator 1) , The Bcl-family of genes are proapoptotic molecules (eg, Bax and Bad)
And apoptosis inhibiting molecules such as, but not limited to, Bcl-2 and Bcl-x ₁ . See Hetts et al., Supra.

Multiple caspases upstream of caspase-3 are inhibited by viral proteins such as cowpox, CrmA, and baculovirus, and p35, synthetic tripeptides and tetrapeptides specifically inhibit caspase-3 ( H above
etts). Therefore, the arrays and subarrays are useful for determining the regulation of gene expression in response to these agents.

This array is also useful for obtaining a series of human (or other animal) orthologs for the drug discovery, treatment, diagnosis and other uses disclosed herein. Can be used. This subarray can be used to tailor a corresponding human (or other animal) subarray associated with a specific biological function. Thus, methods are provided for obtaining sets of genes from other organs, the sets being associated with, for example, diseases or developmental disorders.

In preferred embodiments of the invention, the arrays and subarrays disclosed herein are "microarrays". The term "microarray" is intended to indicate an array of nucleic acid sequences on a chip. This involves in situ synthesis of the desired nucleic acid sequence directly on the chip material, or addition of previously chemically synthesized nucleic acid sequences or nucleic acid sequences produced by recombinant DNA methods on the chip material. Including that. In the case of recombinant DNA methods, the nucleic acid may include the entire vector containing the desired insert (eg, phage and plasmid), which may be PCR cloned, cDNA synthesized from mRNA, to avoid degradation. It is extracted from mRNA etc.

A review of the state of the art in the series of techniques for the production of nucleic acid microarrays, in various forms and examples of applications dealing with biological problems, can be found in Nature Gene.
tics, 21 Supplement, January 1999. These topics include molecular interactions on microarrays and cDNA.
A. Expression Profiling Using Microarrays, Microarray Generation and Reading, High Concentration Synthetic Oligonucleotide Arrays, Sequence and Mutation Analysis Using Oligonucleotides, Use of Microarrays in Drug Discovery and Drug Development, Gene Expression Informatics, and Populations Examples include the use of arrays with genes. Various microarray substrates, methods of treating the substrate to add the nucleic acid onto the substrate, processes for hybridizing the nucleic acid on the substrate with an external nucleic acid sample, methods for detection, and expression using specific algorithms Methods for analyzing data are widely disclosed in the art. The references disclosing various microarray technologies are listed below.

Lashkari et al. (1997) “Yeast Microarrays f.
or Genome Wide Parallel Genetic G
ene Expression Analysis "Proc. NatL Ac
ad Sci. 94: 13057-13062; Ramsay (1998) "D.
NA Chips: State-of-the-Art "Nature Bio
technology 16: 40-44; Marshall et al. (1998) ".
DNA Chips: An Array of Possibilities "
Nature Biotechnology 16: 27-31; Woodick
a et al. (1997) "Genome-Wide Expression Moni.
touring In Saccharomyces Cerevisiae "N
ature Biotechnology 15: 1359-1367; Sou
then et al. (1999) "Molecular Interactions"
On Microarrays "Nature Genetics 21 (1)
: 5-9; Dugganra (1999) Nature Genetics 21.
(1): 10-14; Cheung et al. (1999) "Making and R.
"Eading Microarrays" Nature Genetics21
(1): 15-19; Lipshutz et al. (1999) "High Densi.
ty Synthetic Oligonucleotide Arrays "
Nature Genetics 21 (1) 20-24; Bowtel (19
99) Nature Genetics 21: 25-32; Brown et al. (19).
99) "Exploring the New World of the G
Ename with DNA Microarrays "Nature Ge
netics 21 (1): 33-37; Cole et al. (1999) "The Ge.
NETICS OF CANCER-A 3D Model "Nature
Genetics 21 (l): 38-41; Hacia (1999) "Res.
equaling and Mutual Analysis Us
ing Oligonucleotide Microarrays "Natu
reGenetics 21 (l): 42-47; Debouuck et al. (1999).
) "DNA Microarrays in Drug Discovery
nd Development "Nature Genetics 21 (l)
: 48-50; Bassett, Jr. Et al. (1999) "Gene Expre
session Informations-It's All In Your
Mine "Nature Genetics 21 (l): 51-55; Cha.
Kravarti (1999) "Population Genetic--M
aking Sense Out of Sequence "NatureGe
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sing Genetic Information with High-D
"ensity DNA Arrays" Science 274: 610-61.
4; Lockhart et al. (1996) "Expression Monitori.
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Oligonucleotide Arrays "NatureBiotech
14: 1675-1680; Tamayo et al. (1999) "In.
terpreting Patterns of GeneExpressio
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and Application to Hematopoietic Diff
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07-2912; Eisen et al. (1998) "Cluster Analysis.
and Display of Genome-Wide Expressi
on Patterns "Proc. Natl. Acad Sci. 95.14
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is for Gene Expression Arrays "Nature
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information "Proc. Nall. Acad Sci. 95: 1190
9-11914; U.S. Pat. No. 5,837,832, U.S. Pat. No. 5,861,2.
42; WO 97/10363.

In this case, the microarray comprises nucleic acid sequences on the Biodyne B filter. However, media that are well known and available to those of skill in the art to which nucleic acids can be added in a suitable manner so that they can hybridize are included in the present invention. This includes, but is not limited to, any of the membranes disclosed in the above references, which are incorporated herein as well as other membranes commercially available. These membranes include nitrocellulose-1, trocellulose-1 for support, and Biodyne A (these are neutral charged nylon membranes suitable for Southern transfer and Southern blotting procedures) (all in Life Tec).
commercially available from Hnologies), but is not limited thereto.

EXAMPLE Summary [0284] Rat cerebellar granule neurons (CG) induced by potassium (K ⁺ ) desorption.
Programmed cell death (PCD) in N) has been shown to depend on de novo RNA synthesis. The present invention characterizes the transcriptional components of CGN programmed cell death using a custom-built brain-biased cDNA array representing 7,000 different rat genes. Consistent with carefully integrated mRNAs, the profile of 234 differentially expressed genes reveals genes involved in direct physiological responses, including cell-cell signaling, nucleic acid recognition, apoptosis, and differentiation. It is divided into different temporal groups (early, early, intermediate, and late). A series of 64
Genes (including 22 novel genes) were regulated by K ⁺ elimination and kainate treatment. Human hormones have been isolated for eight of these novel regulated genes. The sequences of these human hormones are SEQ ID NO: 1 (human NARC9
B), 2 (human NARC8B), 3 (human NARC2A), 4 (human NARC1
6B), 5 (human NARC10C), 6 (human NARC1C), 8 (human NAR)
C1A), and 10 (human NARC25). Therefore, array technology has been used to extensively characterize physiological responses at the transcriptional level and identify novel genes induced by multiple models of programmed cell death.

[0285]   (background)   In neurons, programmed cell death is an essential component of neural development (
Jacobson et al., 1997; Pettmann and Henderson (
1998); Pettmann and Henderson (1998) Neur.
on20: 633-747), and this is associated with many forms of neurodegeneration
(Hetts (1998) Journal of the America
n Medical Association 279: 300-307). small
In the brain, granule cell development occurs postnatally. The final number of neurons is the cell
Further processes like division and processes like target related programmed cell death.
Present the combined effects of the two. High concentration (25 mM) extracellular potassium (K ⁺ ) -Induced depolarization leads to the survival of cerebellar granule neurons (CGN) in vitro.
Facilitate. High K⁺CGN maintained in serum-containing medium containing low K⁺(5
(mM) containing programmed cell death when switched to serum-free medium
(D'Mello et al. (1993) Proc. Natl. Acad. Sci. US
A 90: 10989-10993; Miller and Johnson (19.
96) Journal of Neuroscience 16: 7487-
7495). The programmed cell death that occurs is due to de novo inhibitors of RNA synthesis.
Having a transcription component that can be blocked (Galli et al. (1995) Jo
urnal of Neuroscience 15: 1172-1179;
Rabini Schulz and Klockgether (1996) Journal
  of Neuroscience 16: 4696-4706). Traditionally
, Regulation of a limited number of specific genes, Northern nucleic acid hybridization (
For example, PTZ-17, Roschier et al. (1998) Biochemica.
l and Biophysical Research Communica
tions 252: 10-13), reverse transcription polymerase chain reaction (RT-PC).
R; For example, c-jun, cyclophilin, cyclin D1, c-fos or
And caspase (Miller et al. (1997) Journal of Cell
  Biology 139: 205-217) and in situ hybridisation.
Zation (eg, RP-8; Owens et al. (1995) Developm).
using the dental Brain Research 86: 35-47),
It was characterized during programmed cell death of CGN.

High density cDNA arrays have been successfully used to perform genomic systematic (ge
have characterized non-wide mRNA expression (Lashkari et al. (1
997) Proc. Natl. Acad. Sci. USA 94: 13057-
13062; Woodicka et al. (1997) Nature Biotechno.
15: 1997). In higher eukaryotes, the strategy was to an array of as many sequences as possible from a known gene (from an expressed sequence tag (EST)) or from an uncharacterized cDNA from a library (Bowtel ( 1999) Nature Genetics 21:
25-32; Duggan et al. (1999) Nature Genetics 2
1: 10-14; Marshall and Hodgson (1998) Natu.
re Biotechnology 16: 27-31; and Ramsay.
(1998) Nature Biotechnology 16: 40-44).
. Cell cycle regulation (Cho et al. (1998) Molecular Cell 2: 6.
5-73, and Spellman et al. (1998) Mol. Biol. Cell
． 95: 14863-14868), fibroblast growth control (Iyer et al. (199
9) Science 283: 83-87), metabolic response to growth medium (De
risi and Brown (1997) Science 278: 680-68.
6), and germ cell development (Chu et al. (1998) Science 282: 69.
Global RNA regulation during cellular processes, including 9-705), has been monitored over time using arrays. The gene expression programs described in these studies show a correlation between normal function and enhanced expression, and also
Provides a comprehensive and dynamic picture of the processes involved (Brown and Botst
Ein (1999) Nature Genetics 21: 33-37). For the cellular process of programmed cell death, DNA chips have been used to identify 12 known genes that are differentially expressed between two states (etoposide treated and untreated cells) (Wang). Et al. (1999) FEBS Le
ters 445: 269-273).

A genomic systematic approach for comprehensive characterization of transcriptional components of rat CGN programmed cell death and for identification of novel neuronal apoptotic genes consists of both known rat cDNA and novel rat DNA. Requires an array. We have identified over 300 genes that are known markers for central nervous and / or programmed cell death, as well as the rat precortex.
differentiated P without tal cortex) and nerve growth factor (NGF)
A death-enriched clone set of brain-biased and programmed cells was constructed by aligning approximately 7300 ligated ESTs from two cDNA libraries from C12 cells. did. We monitored the expression of genes reproducibly and simultaneously at 1, 3, 6, 12 and 24 hours after K ⁺ withdrawal. Then, we
To identify the cellular processes driven by CGN programmed cell death at the NA level, genes regulated by time course expression patterns were grouped. In particular, we have found that transcription factors, Bcl-2 family members, caspases, cyclins, heat shock proteins (HSPs), inhibitors of apoptosis (I
AP), growth factors and receptors, other signaling molecules, p53, superoxide dismutase (SOD) and other stress response genes. It was Finally, we compared the time course of regulatory genes induced by K ⁺ withdrawal in the presence or absence of serum with the time course of regulatory genes induced by glutamate toxicity. Thus, we have identified a restricted set of related genes that are regulated by multiple models of programmed cell death in CGN.

Results Construction and Confirmation of Brain-Energized cDNA Microarrays To characterize the transcriptional constituents of neuronal apoptosis in rat cerebellar granule neurons, we called the Smart Chip ^™ I. c
A DNA array was constructed. It contains the major rat brain genes. FIG. 1 is a schematic diagram of the construction of this microarray. Two cDNA libraries were cloned from rat precortex and nerve growth factor-depleted PC12 cells to enrich for cDNA expressed in one in vitro model of central nervous system and neuronal apoptosis. The expressed sequence tag (EST) from the 5'end was assigned to 8,304 clones in the cortical library and 5 in the PC12 library.
, 680 clones were identified. These 13,984 ESTs were identified by Basic Local Alignment to identify ESTs with overlapping sequences.
ent Search Tool (BLAST) sequence comparison analysis (Altsch
1 et al. 1990) and cloned into 7,399 single sequence clusters. One representative clone was selected from each of 7,296 single sequence clusters and prepared for PCR amplification using a robotized sample processor. In addition to ESTs, PCR templates were prepared for 289 known DNA sequences, using negative controls, genes with known functions in the CDN and / or during programmed cell death, and differential display. And previously identified genes when regulated by CGN programmed cell death (data not shown). To check the accuracy of the set of array elements, a robotized sample processor was used to randomly select 212 clones for sequencing. 10 clones yielded poor sequence. The remaining 202 matched their seed sequence (data not shown), which implies 100% fidelity in sample tracking.

A 20 nl sample volume from each of the 7584 PCR products was aligned onto a nylon filter at about 64 / cm ² using a pin robot. Aligned D
The NA element was denatured and covalently attached to a nylon filter for use in inverted Northern nucleic acid hybridization experiments. In a representative experiment, “radiolabeled RNA” (1 μg of poly A RNA radiolabeled by ³³ P-dCTP incorporation during cDNA synthesis) was hybridized to triplicate arrays after RNA hydrolysis. Subsequently, the filter was washed and exposed to a phosphoimage screen. Gene expression was quantified for each array element by digitizing the phosphoimage-capture hybridization signal intensity. FIG. 2 illustrates that the coefficient of variation between the three hybridizations is an average of less than 0.2 for genes whose intensity exceeds the threshold of 30-40 units. From control experiments, this threshold reached a copy number of less than 1 in 100,000 when the in vitro transcribed DNA spiked slowly into the sample (data not shown).

Tissue distribution of brain-energized Smart Chip ESTs To characterize the brain-energized cDNA array and to identify as brain-specific genes as possible, 10 different radiolabeled RNAs from normal rat tissues were labeled with S.
Hybridized to the Mart Chip. Radiolabeled rat brain RNA produced more hybridization signal intensity for most brain-biased array elements as compared to heart, kidney, liver, lung, pancreas, skeletal muscle, smooth muscle, spleen and testis. . After data normalization and averaging between replicates, the threshold of detection was determined for each experiment and tabulated for the number of genes detected for each tissue (Figure 6).
. Most (6127 out of 7926) but not all ESTs were detected in at least one of the profiled tissues. The number of genes detected in the brain was the highest. The 582 gene appeared to be brain-specific, as defined by detection above the threshold for the brain but below the threshold for all nine other tissues.

(Characterized by Transcriptional Profiling on the Smart Chip,
CGN KCl / serum withdrawal physiology Using brain-enhanced, programmed cell death nucleic acid-enriched Smart Chips, global mRNA expression was induced by KCl / serum withdrawal in primary CGN cultures. Profiled over a time course of cell death. Transcription-dependent CG
There was less than 30% survival after 24 hours of withdrawal when N-programmed cell death was coordinated and quantified by cell counting (data not shown). RNA samples (referred to as "treated") were prepared from 5% serum and 25 mM KCl to 5
8 day old CGN were isolated 1, 3, 6, 12 and 24 hours after conversion to serum-free medium with mM KCl. As a control, 5% serum / 25 mM K
The C1 medium was replaced, and "sham" RNA was isolated after 1, 3, 6, 12 and 24 hours.

The mean coefficient of variation for gene expression intensity during triplicate hybridization was less than 0.2, so at least 3 during the time course.
A double regulated gene (790 of 6818 detected; data not shown),
Further addressed. Using a hierarchical culture algorithm (see Experimental Procedures), regulated genes were ordered based on their gene expression pattern across 10 experimental points (5 time points, sham and processed). (Figure 3)
). The phylogenetic tree of Figure 3 shows the hierarchy of associations between gene expression profiles. The first major branch point sequesters these genes controlled by sham treatment (first 5 rows) and they are controlled only by KCl / serum-withdrawal treatment (last 5 rows). . Most of the genes (556) were controlled by sham treatment. These genes include trkA, PSD-95, SV2A, and VAMP1 and were challenged at t = 0 with unconditioned medium, resulting in serum-add- back).

FIG. 3 shows that the expression of 234 programmed cell death-inducing genes is controlled by KCl / serum withdrawal only, not by serum-titration in sham experiments. Their coefficient of variation in expression levels over 5 serum-titration experiments was less than 20%. Since serum-titration experiments were not discriminatory for these genes, the serum-titration data were averaged over one control data set for clustering using the KCL / serum withdrawal time course. There are four distinct temporal control classes: earliest (peaking at 1 hour followed by rapid decline), early (peaking at 3-6 hours), middle (6-
12 hours peaking) and late (24 hours upregulation). Almost all of the immediate early genes encoded proteins with known roles in secretion and synaptic vesicle release. Examples of these proteins include synaptodagumin, synaphin, NSG-1, calcium calmodulin-dependent kinase II, synapsin, complexin (c
oplexin), LDL receptor, and fodrin (Fig. 7).
. Histones 1, 2A and 3 were classified in the early class. Metaphase genes included several known genes that are induced by programmed cell death or stress. These include caspase 3, mammalian oxy R homologues, cytochrome c oxidase and protein phosphatase Wip-1. Functions encoded by late genes include inhibitory neurotransmission (GAB, GABA-A receptor, GABA transporter), cell adhesion (nexin, basement membrane protein 40, phosphacan, rat GRASP), and excitatory neurotransmission. Down regulation (glutamate transporter, sodium-dependent glutamate /
Aspartate transporter), leukotriene metabolism (dithiolethione (dithiol)
Ethione) -induced NADP-dependent leukotriene B4 12 hydroxydehydrogenase, leukotriene A-4 hydrolase), protein stabilization (cysteine proteinase inhibitor cystatin C, N-α-acetyltransferase GaBP2, elongation factor 1-γ, APG-1) and ionicity. Equilibrium and cell volume (SLC12A integral membrane protein transporter) are included. Based on four distinct waves of gene expression, the major transcriptional responses observed for KCl / serum-withdrawal included an early upregulation of synaptic vesicle release / recycling, followed by upregulation of histone biosynthesis, This is followed by various components of programmed cell death regulation and stress-responsive signaling, and finally, the upregulation of multiple survival mechanisms. Apparent changes in transcription also appear to reflect the changes in the cell populations involved. Because late mRNA can be a marker for neuronal and non-neuronal cells, as it survives KCl / serum-withdrawal at 24 hours. Another contributing factor may be present in the two populations of moribund neurons that respond with different kinetics to serum viral KCl withdrawal, as described by other groups.

Neuronal Apoptosis Control Candidate (NARC) Controlled by Multiple Models of Programmed Cell Death 112 novel ESTs were significantly regulated by KCl / serum-withdrawal in rat CGN (data not shown). Some showed similar expression profiles over KCl / serum-withdrawal and serum-modulation for genes with known function during programmed cell death (eg caspase 3). The time-related expression of these novel genes may reflect the related functionality with caspase-3. Because they probably share common RNA regulatory elements, including elements that control initiation, extension, processing and / or stability.
The apparent coordinated transcriptional up-regulation of synaptic vesicle release / recycling likely reflects the physiological response to a near arrest of synaptic transmission that may or may not contribute to the programmed cell death pathway. To further aid in distinguishing genes that are specifically regulated in response to programmed cell death, we studied CGN programmed cell death induced by glutamate (stimulatory neurotransmitter) toxicity. In addition, the effect of KCl-withdrawal alone on gene expression was tested. This was done under defined media conditions that minimized the effect of serum on sham and treated samples.

Rat CGN from 7 day old pups were isolated as described above and "high" 1
Plated in basal medium Eagle containing 0% dialyzed fetal bovine serum and "high" 25 mM KCl. "Low" 0.5% serum and high KC after 2 days of culture
The medium supplemented with 1 was replaced with neurobasal medium. The KCl concentration was converted to 5 mM for the treated samples to initiate KCl-withdrawal on day 8. The same low serum, high KCl neurobasal medium was replaced under control to minimize gene induction by high serum. For glutamate toxicity experiments, cells were treated for 30 minutes in rock medium without sodium with or without 100 μM kainate for each treated sample or control.

1, 3, 6 and 12 hours after KCl-withdrawal and 2 after kainate treatment,
MRNA was subjected to expression profiling analysis on Smart Chip I after isolation from treated and control samples after 4, 6 and 12 hours. Figure 4 shows that CGN is KCl / plasma-withdrawal, KCl-withdrawal only,
Or exemplifies changes in gene expression that occur over time induced by kainate treatment to undergo programmed cell death. In the scatter plot, due to differential expression, a large number of regulated genes migrated away from the slope 1 line when the withdrawn (W) or treated (T) samples were compared to the control (C). . Cells mock-treated for KCl / serum-withdrawal clearly responded to basal medium serum-modulation, whereas mock samples for KCl withdrawal only and kainate treatment did not respond to conditioned neural basal medium modification. . MRNA of thousands of genes
Profiling across levels provided a clear indicator of changes in overall cell physiology.

In general, the apparent changes in gene expression were less potent in cells cultured on neurobasal medium. The number of genes that detected the above threshold was similar for all three conceptual schemes (6634, 7017 and 6818, which were KCl withdrawal, kainate treatment, and KCl / plasma withdrawal, respectively) (data not shown). .. Nevertheless, the number of genes that were at least 3-fold regulated during KCl-withdrawal and kainate treatment was discussed above for KCl / plasma withdrawal 790
There were only 156 and 167, respectively, compared to (data not shown).

A hierarchical clustering algorithm was used to align the regulated genes based on their gene expression patterns across all CGN programmed cell death schematics investigated. Twenty-six individual profiling experiments, in duplicate or triplicate, were given 5 serum-modulation time points, 5 KCl / plasma-withdrawal time points, 4 time points for sham and KCl-withdrawal, and 4 each for sham and kainate treatments. MRNA isolated from time points was used across the 7584 rat gene on the Smart Chip I.

FIG. 4 shows expression clusters created by one stepwise clustering algorithm. This insert shows a particular group of genes with similar expression patterns. This group contains genes known to be regulated in programmed cell death (eg, Caspase 3 and Wip1 and other nucleic acid sequences on the array that were not previously known to be regulated). Those sequences that met the specified criteria were referred to as "neuronal apoptosis control candidates (NARC)". The criteria for naming such genes were based on specific expression criteria as shown in FIG. A nucleic acid sequence with an expression pattern similar to genes known to be involved in apoptosis was designated as the NARC sequence.

(Confirmation of gene expression by RT-PCR) The reproducibility in the transcription profiling experiment is very high (mean CV <0.2.
) Regulates gene expression of known genes and novel genes by using quantitative quantitative RT-
Confirmed by PCR. Several NARs whose expression is involved in programmed cell death (when hybridized to sequences on a chip) using the rat CGN model system
Expression of the C gene was independently confirmed. Reverse transcription assisted PCR was performed using NARC1-7, 9
, 12, 13, 15 and 16 were evaluated. Experimental samples underwent KCl withdrawal treatment. Control samples represent untreated cells. PCR reactions were performed with 10, 5, 2.5, 1.3 and 0.7 ng total RNA each
Included. The RT-PCR protocol is disclosed in the exemplary materials herein. NARC 1, 2, 4, 5, 7, 9, 12, 13, 15 and 16 are all
It showed significantly increased expression 3-6 hours after KCl withdrawal. The above name "N" is
It is an abbreviation for the acronym "NARC", which is an abbreviation for "neuronal apoptosis control candidate" described in the Examples section.

Regulation of NARC1 and NARC2 in vivo during cerebellar development Two novel neuronal apoptosis control candidates (NARC1 and NARC2).
Was confirmed by in situ hybridization and was shown to be coordinately upregulated with caspase-3 during postnatal development when increased apoptosis was associated with synaptic sclerosis in the cerebellum (not shown).

Experimental Procedure BLAST Sequence Comparison Analysis Two cDNA libraries, cDNA extracted from rat precortex (8,304 clones) and NGF-deficient differentiated PC12 cells (5,680 clones).
The ESTs determined for the 5'end of A ranged in sequence length from 100 to 1000 nt and averaged 500 nt (data not shown). Sequence comparison B
This was done using LAST (Altschul et al. 1990). Sequential matches defined sequence clusters. Large clusters were manually examined to remove obvious chimeras. From the inserted 13,984 sequences, this analysis shows that
Nine singletons and 1,620 large clusters were identified (data not shown). Most of the 5'clones were selected from the large cluster. Due to the lack of clones for the two 96-well microtiter plates, 7,296 of the 7,399 identified were Smart Chip ^™ I.
Selected for.

CDNA Microarray Constructs Using the RSP150 robotized sample processor (Tecan AG, Switzerland), microbial cultures of individual EST clones from two libraries were prepared.
296 Smart Chip I clones spanning 76 plates to 144 of 9
A 6-well microtiter plate was integrated from cross-linked 13,792 clones. To prepare the template for the array elements, the cDNA insert was amplified by PCR using oligonucleotide primers upstream of the cloning site and specific to the vector sequences of the granules. Ethanol precipitation and centrifugation (1-1
After 0 mg / ml), the array element template was treated with 3 × SSC (1 × SSC: 150).
Resuspended in mM sodium chloride, 15 mM sodium citrate (pH 7.0)). A sample volume of 20 nl from each template was used with a 96-well format pin robot (THOR) at about 64 / cm ² with a nylon filter (Biod.
yne B, Gibco BRL Life Technologies, Ga
Itersburg, MD). After the filter is dried, the aligned DNA is denatured in 0.4M sodium hydroxide and 0.1M Tr
Neutralized with is-HCl (pH 7.5), rinsed in 2xSSC and dried completely.

Array Hybridization Rat poly A ⁺ RNA was labeled with Clontech (Palo) for organ details.
Purchased from Alto, CA) or RNA STAT-60 ^™ (Tel
-Test, Inc. , Friendswood, TX) was used to isolate total RNA from cultured CGN and then Oligotex ™ (Qiagen).
, Inc. , Chatsworth, CA). Re-annealed 1 μg mRNA and 1 μg oligo (dT) ₃₀ with 0.5 mM of each deoxyribonucleotide (dATP, dGTP and dTTP) and 100.
Incubated for 30 minutes at 50 ° C. using SuperScript ^™ as recommended by Gibco in the presence of μCi α ³³ P-dCTP (2000-4000 Ci / mmol; NEN ^™ Life Science Products, Boston, MA). . Chroma Spin ^TE +30
After purification on a column (Clontech), 10 μg of poly (dA) _{> 200} and 10 μg of rat Cot-1 DNA (Britten et al. (1974) Methods in Enzymology 29: 263-4) were purified by labeling.
(Prepared as described in 18). 2 x 10 ⁶ cpm
/ Ml of the annealed cDNA mixture in a pre-annealing solution containing 100 mg / ml sheared salmon sperm DNA in 7% SDS (sodium dodecyl sulfate), 0.25 M sodium phosphate, 1 mM ethylenediaminetetraacetic acid and 10% formamide. Added to array filter. Rotating incubator (Ro
After hybridization overnight at 65 ° C. in bbins Scientific, Sunnyvale, CA), the array filter was 2 × at 22 ° C.
SSC, 1% SDS twice for 15 minutes at 65 ° C. 0.2 × SSC, 0.5% S
Washed twice in DS for 30 minutes and twice at 22 ° C. in 2 × SSC for 15 minutes. The array filter was then dried and exposed to a phosphoimage screen for 48 hours. This radioactive hybridization signal was given to Fuji.
Captured using a BAS 2500 Phosphoimager and Array V
Iion ^™ software (Imaging Research Inc.,
(Canada). Array hybridizations for organ details, withdrawal of CGN KCl alone, and CGN kainate treatment experiments were performed in triplicate; for CGN KCl / serum-withdrawal, these were done in duplicate.

Transcriptional Profiling Data Analysis For replicate array hybridization, the distribution of signal intensity across all rat genes was normalized to a median of 100. Duplicate measurements were averaged and the coefficient of the variable (CV; standard deviation / 3 triplicate mean or absolute difference / 2)
The average of runs) was determined for each gene. The threshold of detection was selected for each hybridization experiment by graphing the moving averages with CV vs. average gene expression intensity (using a window of 200) (FIG. 2).
. This threshold was defined as the intensity at which relatively low intensity exhibits an average CV of greater than 0.3. For the majority of experiments, this threshold ranged from 10-40, and the number of genes detected above the threshold ranged from 70% -95%.

CGN Cell Culture CGN was prepared from rat pups at several days of age as previously described (John).
Son and Miller (1996) Journal of Nurosci
ence 16: 74877-7495). Briefly, cerebellum was isolated and the meninges and blood vessels were dissected under an anatomical microscope. The separated cells were treated with 25 mM K
Cl, 10% dialyzed fetal bovine serum (Summit Biotechnol
ogy lot number 04D35, Ft. Collins, CO), 100 U / m
Plated at a density of 2.3 × 10 ⁵ cells / cm ² in Eagle's basal medium (BME; Gibco) supplemented with 1 penicillin and 100 μg / ml streptomycin. Affidikoline (Sigma, St. Louis, MO
) Was added to the cultures at 3.3 μg / ml 24 hours after the initial plating to reduce the number of non-neuronal cells to less than 1-5%.

For KCl / serum withdrawal experiments, after a few days in culture, treated cells were switched to 5 mM KCl, BME, serum-free, while mock underwent media change. After withdrawal 2
By 4 hours, less than 30% of the cells were viable when assayed by Hoechts cytometry (data not shown). This apparent cell death is 2
Can be rescued by actinomycin D at μg / ml (data not shown).

For KCl withdrawal only and kainate treatment experiments, on day 2 of culture, the medium was 25 mM KCl, 0.5% dialyzed fetal calf serum, B27 application (Gibco), 0. 5 mM L-glutamine (Gibco), 0.1 mg / m
l AlbuMAXI (Gibco), 100 U / ml penicillin, 100μ
Neurobasal medium (Gibco) supplemented with g / ml streptomycin and 3.3 μg / ml afridicolin was changed. On day 7, remove KCl by 5 mM
Was started by changing to medium containing KCl, while sham received 25 mM. Twenty-four hours after withdrawal, 40% of the cells were viable when assayed by Hoechts cytometry (data not shown). As previously mentioned,
Glutamate toxicity was induced by exchanging the medium for 30 minutes with 5 mM KCl, 100 μM kainic acid (Sigma) in rock buffer without sodium, while sham did not receive kainic acid. Et al (1996
) Neuroscience 74: 675-683). After 30 minutes, the supplemented neural basal medium was replaced. By 12 hours post withdrawal, 30% of cells were viable when assayed by Hoechts cytometry (data not shown). K
Cl withdrawal-induced cell death was rescued by actinomycin D, whereas kainic acid-induced cell death was not.

Expression Data Cluster Algorithm After normalization and averaging of KCl / serum recovery data, 790 genes were submitted for 10 time points (5 treatments, and 5 shams) passed the following criteria: 1
． Detection, maximum intensity is greater than 30; 2. 2. Noise filter, difference between maximum and minimum intensities greater than 30, and 3. Adjustment of at least 3,
Fold induction between maximum and minimum intensity (data not shown). The hierarchical raster was ordered based on Euclidean distance. 234 of the 790 genes that passed the significance filter above were not regulated in controls based on a CV of <0.2 for all 5 control time points (data not shown). ).

RT-PCR Oligonucleotide primer sequences specific for each EST verified by RT-PCR were selected from the quality sequence region, and Bresla
uer et al. (1986) Proc. Natl. Acad. Sci. USA 83:
PrimerSele based on DNA stability measurements by 3746-3750
Designed to obtain a melting temperature of 55-60 ° C, as estimated by ct software (DNASTAR, Inc., Madison, WI). Str
atagena Opti-Prime ^™ Kit (La Jolla, CA)
) Was used to determine the optimal RT-PCR amplification conditions for each primer pair. RT-PCR reactions on 2 fold serially diluted CGN programmed cell death cDNA were set up using a Genesis RSP 150 robotic sample processor and incorporating optimal buffer conditions for each primer pair. Was done. All operated robots contained primers specific to the housekeeping gene to control the daily differences in the cDNA template dilutions. The number of cycles was adjusted to obtain a linear range of amplification by comparing the amount of product made from serially diluted template, as assessed by agarose gel electrophoresis.

(Preparation of Array on Nylon) (I. Superscript II Reverse Transcript
Procedure for producing labeled first-strand cDNA using ase) 1.10 mL (100 mCi) of 33P α-dCTP was added to SpeedVac
Dried by.

[0312]   2. The following ingredients were mixed in separate tubes:     1.0 ug poly A + RNA or 10 ug total RNA     1 ug / uL oligo-dT (30) was added to 1 uL     xuL DEPC-H2O, up to 10 uL. The above sample was heated to 70 ° C. for 4 minutes and then placed on ice.

3. Remove 8 uL from oligo / RNA mixture (# 2) and dry 3
Used to resuspend P3. The following components were added to this reaction: 4 uL of 5x First Strand Buffer (Superscr
2 μL of 100 mM DTT 1 uL of 10 mM dAGT-TP 1 uL of 0.1 mM cold dCTP 1 uL of Rnase inhibitor 1 uL of Superscript II RT The reaction was incubated at 50 ° C. for 30 minutes.

[0314] 4. After the incubation, 2 uL of 0.5 M NaOH and 2 uL of 10 mM EDTA were added. Heat the reaction to 65 ° C. for 10 minutes to remove RNA
The template was disassembled.

[0315]   5. The volume was brought to 50 uL (ie, 26 uL H2O was added).

6. One Choma-Spin + TE for all prepared probes
30 columns (Clontech, # K1321) were prepared.

[0317]       a. Bubbles were removed from this column.

B. The desorption end point (break-away end) of this column is removed,
The column was then placed in an empty 2 mL tube and spun at 700 g for 5 minutes (in Eppendorf 5415C "3.5").

C. The column was removed and the flow through was discarded. The column was placed in a clean tube. The probe was slowly added to the center of the column base without disturbing this matrix so that liquid did not contact the sides of the column and did not flow down the edges of the column wall. The probe was eluted by spinning the column as above.

[0320]   (II. Hybridization)   1. The hybridization chamber was preheated to 65 ° C.

2.10 mL of 10% Formamide Church Buffer was added. It was placed in the hybridization chamber for about 15 minutes.

3. The sheared salmon sperm DNA was denatured at 95 ° C. for 5 minutes, placed on ice and then added to the hybridization mixture at a final concentration of 100 ug / mL. Prehybridization was for 1.5 hours.

The amount of probe required to reach 2 × 10 ⁶ cpm / mL for 4.10 mL was calculated.

5. Cott Annealing Reaction (per bottle)
Was as follows: Rat probe on Rat Filters: 10 ug poly dA (> 200 nt) 10 ug rat Cot 10 DNA 25 uL 20 × SSC probe + water up to 100 uL Rat Probes mouse probe: 10 ug poly. dA (> 200 nt) 10 ug mouse Cot 1 DNA 25 uL 20x SSC probe + water up to 100 uL For this prehybridization 5 ug rat Cot 10 DN
Human probe on Human Filters with A also added: 10 ug poly dA (> 200 nt) 10 ug human Cot 1 DNA 25 uL 20 × SSC probe + 100 μL water heated this probe to 95 ° C. and then this probe 65 Pre-anneal at 1.5 ° C. for 1.5 hours.

6. The probe was added to the pre-ived filter (directly in solution, not on the filter) and allowed to hybridize for approximately 20 hours.

[0326]   (III. Washing)   1. The probe was removed.

2. Three quick washes were performed at 65 ° C. using preheated 2 × SSC / 1% SDS (washing can be done in roller bottles).

3. Two washes of 15 minutes each with preheated Takashu and Lingent wash buffers: 0.5 × SSC for hybrid wash, 0.5 × SSC for 0.1% SDS regular wash, 0.1% SDS 0.1 × SSC, 0.1% S for very high states and lingent washes
DS.

[0329] After a high stringency wash, the filters were rinsed in large square Petri dishes without 2xSSC, SDS. Due to the experiments in which many filters are used, the 2 × SSC is frequently replaced, resulting in residual SDS on this filter.
Did not remain.

5. The filter was removed from the 2 × SSC and placed on Whatman filter paper. The filter was calcined at 85 ° C for over 1 hour. The screen was protected against any moisture. The filter was placed on a blank phosphoimager screen. No yellow phosphoimager screen was used. Because they cannot respond linearly to exposure. The screen disappeared on the lightbox for over 20 minutes.

6. Blots were exposed to the screen for at least 48 hours or as needed.

(IV. Scanning Filter on Fuji Phosphorimager) Gradation 16 bits, resolution 50 m, dynamic range S40
00, selective reading and activation image gauge. The image was saved to your hard drive.

Appendix I: 10% Formamide-Church Buffer: 59.6 mL Water 70 mL 20% SDS 50 mL 2M NaPO4 pH 7.2 20 mL Ultrapure Formamide 0.4 mL 0.5M EDTA pH 8.0 Add the above components to water. , Mixed and filtered through a 0.2 um filter.

[0334]   (RT-PCR protocol)   I. The following components were used for one PCR reaction mixture:     28 ul of 5x First Strand Buffer     14ul of 0.1M DTT     4 ul dNTPs (20 mM)     7 ul Rnase inhibitor     7ul of Superscript II This buffer may be stored at -80 ° C for 3 months.

II. Total RNA was reverse transcribed as follows: 1.4 ug total RNA (DNAse treatment) 14 ul of random primer (50 ng / ul-Gibco) Water was added up to 60 ul. Incubate this mixture at 70 ° C. for 10 minutes,
It was then placed on ice for 2 minutes. 60 ul of RT reaction mixture was added. Incubation is carried out at room temperature for 10 minutes, then at 50 ° C. for 30 minutes, then 90
It was carried out at 0 ° C for 10 minutes. The sample was diluted with 480 ul of water, yielding 10 ng per 5 ul.

III. PCR reactions were performed with the following components: 5 ul 4x PCR buffer 5 ul cDNA (10 ng / 5 ul) 5 ul 1 uM primer pair 5 ul enzyme cocktail (0.2 ul Hot Start Taq, 1 ul.
2 mM dNTPs, 3.8 ul water.

IV. Cycling was as follows: 95 ° C. for 15 minutes 94 ° C. for 30 seconds 52 ° C. for 30 seconds 72 ° C. for 1 minute Cycles 26-30 times 72 ° C. for 10 minutes 4 ° C. Isolation of cerebellar granule cells In Johnson et al. (1996) J. Am. Neurosci
． 16: 74877-7495.

Induction of apoptosis in neurites induced by kainate is described by Ne
urosci. 75: 675-683 (1996). The procedure shown in this reference is as follows.

The following parameters were checked: (1) Viability of cerebellar granule neurons after recovery of potassium and serum at this point (Hoechst), corresponding to a PCR-based method for differential gene expression.
staining).

(2) Effect of 2 ug / ml actinomycin D on recovery of potassium and serum at 24 h in cerebellar granule neurons; viability by Hoeschst stained cell count.

(3) Time course of kainate-induced cell death for parallel analysis of PCR-based methods for differential gene expression of CGN poly A mRNA.

(4) Time course of kainate-induced apoptosis (30 min exposure) in CGN; analysis by Hoechst cell count.

(5) PCR for differential gene expression in Hoechst count analysis
Time course of potassium recovery apoptosis in CGN in defined media for the base method.

The present invention has been shown and described in detail with reference to its preferred embodiments, but without departing from the spirit and scope of the invention as defined by the appended claims, forms and It will be appreciated by those skilled in the art that various changes in detail may be made therein.

[Sequence list]

[Brief description of drawings]

FIG. 1 illustrates the construction of “Smart Chip ^™ I”. cDNA was cloned from rat frontal cortex and from differentiated PC12 cells deficient in nerve growth factor, a model of programmed cell death, as described in detail in the experimental section. PC12 cells are a rat-derived adrenal cell line that provides a pre-neuronal set that can be differentiated in vitro. Application of nerve growth factor induces the formation of axons and dendritic structures. It serves as a model for neuronal differentiation. When nerve growth factor is withdrawn, the cells undergo programmed cell death (apoptosis). About 300 control nucleic acid sequences (of known function) were added as internal controls and for transcriptional profiling of these cloned cDNA sequences. These sequences are then subjected to BLASTX analysis to determine the correspondence between the cDNA and the known cDNA, and each c
It was determined to which protein family (if any) these proteins encoded by the DNA belong. Using computer analysis, these cDNAs
The A sequence was assembled into unique clusters. Most of these clusters and control genes were gridded on the Smart Chip ^™ I (gr
idled).

FIG. 2 shows the coefficient of variation (standard deviation / mean for triplicate hybridizations) after normalization for each array element, plotted against mean intensity of genes (gene expression intensity). This figure shows the moving averages (with 200 windows) for three different mRNA probes, 3 hour KCl withdrawal, 3 hour control and 6 hour control (see Example and Figures 3 and 4). As typical for all probes, a threshold of 30-40 is exceeded and the coefficient of variation is below 0.2 on average. The inset compares one triplicate hybridization (filter Y) to another triplicate hybridization (filter Z). Each point represents a different gene graphed on the log-log axis, which compares the intensities measured for one filter to the other.

FIG. 3 shows expression clusters over time observed after withdrawal of KCl and serum. Using the hierarchical clustering algorithm, 10 time points (from left to right) (
Cluster genes based on expression patterns passing 1, 3, 6, 12 and 24 hours after KCl / serum replacement (mock) and 1, 3, 6, 12 and 24 hours after KCl / serum withdrawal (treatment)) (See examples). The expression value for each gene was measured based on the number of standard deviations from the mean intensity of each gene passing through all 10 time points. The measured expression values are color-coded so that red, yellow and blue are shown above average intensity, below average intensity and below average intensity, respectively. The correlation between the expression patterns of adjacent genes is shown by the phylogenetic tree of light. Genes regulated by programmed cell death (KCl / serum withdrawal alone) are detailed in B. Typical non-scale (non-scal) with standard deviation error
led) Gene expression bar graphs were sorted in sequence into their four major clusters for expression classes of late effector, metaphase, early and immediate early genes. The regulated genes within each temporal expression class are listed in order of hierarchical clustering in SEQ ID NOs: 1-6, 8 and 106.

FIG. 3B shows expression clusters over time observed after withdrawal of KCl and serum. Using the hierarchical clustering algorithm, 10 time points (from left to right) (
Cluster genes based on expression patterns passing 1, 3, 6, 12 and 24 hours after KCl / serum replacement (mock) and 1, 3, 6, 12 and 24 hours after KCl / serum withdrawal (treatment)) (See examples). The expression value for each gene was measured based on the number of standard deviations from the mean intensity of each gene passing through all 10 time points. The measured expression values are color-coded so that red, yellow and blue are shown above average intensity, below average intensity and below average intensity, respectively. The correlation between the expression patterns of adjacent genes is shown by the phylogenetic tree of light. Genes regulated by programmed cell death (KCl / serum withdrawal alone) are detailed in B. Typical non-scale (non-scal) with standard deviation error
led) Gene expression bar graphs were sorted in sequence into their four major clusters for expression classes of late effector, metaphase, early and immediate early genes. The regulated genes within each temporal expression class are listed in order of hierarchical clustering in SEQ ID NOs: 1-6, 8 and 106.

FIG. 4 shows expression clusters for all CGN programmed cell death models (KCl and serum withdrawal, KCl withdrawal alone, and kainate treatment). Figure 4A
Is a self-organizing map (SOM) algorithm (eg Kohonen, Se
lf Organizing Maps: Springer, Berlin (1
997)), which was used to cluster genes based on expression in 26 experiments (Order: serum added.
ck) 1, 3, 6, 12, 24 hours; KCl / serum withdrawal 1, 3, 6, 12,
24 hours; control for KCl withdrawal 1, 3, 6, 12 hours; KCl
Withdrawal alone 1, 3, 6, 12 hours; control for kainic acid treatment, 2
4, 8, 12 hours; kainic acid treatment, 2, 4, 8, 12 hours; see Examples for experimental details). As shown, the genes were organized into 20 groups using 5x4 geometry. A cluster of 17 programmed cell death-inducible genes (3,3) is highlighted. The inset shows a tile representation of all genes in this (3,3) cluster (red = above average expression, white = average expression, blue = below average expression; tiles are as described above for experimental order). , Each row representing a different array element gene, in the order indicated by the distance from each cluster centroid). Caspase 3 (a gene involved in apoptosis) is part of this array and is shown in the raw value graph (ie relative expression in these 26 experiments); each experiment is represented on the x-axis in turn. The y-axis shows gene expression intensity. Figures 4B, C, D and E show the raw genes plotted for programmed cell death regulation, regulation by KCl withdrawal only, immediate early genes, and representative genes from the class of serum suppressive constitutive expression, respectively. The gene expression intensity is shown. Each panel is
Data for representative members of the cluster (indicated in this gene list by ^* ) are shown, along with a list of genes included in the expression cluster. FIG. 4B shows the raw gene expression intensities for representative genes from the list on the right. This graph shows increased expression with KCl and serum withdrawal and kainic acid treatment. Therefore, genes with these characteristics are termed "programmed cell death regulators." A list of genes with this pattern (on chip) is shown on the right. Known genes include genes that are regulated in apoptosis. FIG. 4C lists genes that show increased expression after withdrawal of KCL or KCL and serum (but after kainic acid treatment). This list includes genes known to be involved in apoptosis. FIG. 4D shows genes that show constitutive immediate early expression. FIG. 4E shows genes that show constitutive expression in the absence of serum. The list on the right indicates that this class contains mediators of programmed cell death.

FIG. 4 shows expression clusters for all CGN programmed cell death models (KCl and serum withdrawal, KCl withdrawal alone, and kainate treatment). Figure 4A
Is a self-organizing map (SOM) algorithm (eg Kohonen, Se
lf Organizing Maps: Springer, Berlin (1
997)), which was used to cluster genes based on expression in 26 experiments (Order: serum added.
ck) 1, 3, 6, 12, 24 hours; KCl / serum withdrawal 1, 3, 6, 12,
24 hours; control for KCl withdrawal 1, 3, 6, 12 hours; KCl
Withdrawal alone 1, 3, 6, 12 hours; control for kainic acid treatment, 2
4, 8, 12 hours; kainic acid treatment, 2, 4, 8, 12 hours; see Examples for experimental details). As shown, the genes were organized into 20 groups using 5x4 geometry. A cluster of 17 programmed cell death-inducible genes (3,3) is highlighted. The inset shows a tile representation of all genes in this (3,3) cluster (red = above average expression, white = average expression, blue = below average expression; tiles are as described above for experimental order). , Each row representing a different array element gene, in the order indicated by the distance from each cluster centroid). Caspase 3 (a gene involved in apoptosis) is part of this array and is shown in the raw value graph (ie relative expression in these 26 experiments); each experiment is represented on the x-axis in turn. The y-axis shows gene expression intensity. Figures 4B, C, D and E show the raw genes plotted for programmed cell death regulation, regulation by KCl withdrawal only, immediate early genes, and representative genes from the class of serum suppressive constitutive expression, respectively. The gene expression intensity is shown. Each panel is
Data for representative members of the cluster (indicated in this gene list by ^* ) are shown, along with a list of genes included in the expression cluster. FIG. 4B shows the raw gene expression intensities for representative genes from the list on the right. This graph shows increased expression with KCl and serum withdrawal and kainic acid treatment. Therefore, genes with these characteristics are termed "programmed cell death regulators." A list of genes with this pattern (on chip) is shown on the right. Known genes include genes that are regulated in apoptosis. FIG. 4C lists genes that show increased expression after withdrawal of KCL or KCL and serum (but after kainic acid treatment). This list includes genes known to be involved in apoptosis. FIG. 4D shows genes that show constitutive immediate early expression. FIG. 4E shows genes that show constitutive expression in the absence of serum. The list on the right indicates that this class contains mediators of programmed cell death.

FIG. 4 shows expression clusters for all CGN programmed cell death models (KCl and serum withdrawal, KCl withdrawal alone, and kainate treatment). Figure 4A
Is a self-organizing map (SOM) algorithm (eg Kohonen, Se
lf Organizing Maps: Springer, Berlin (1
997)), which was used to cluster genes based on expression in 26 experiments (Order: serum added.
ck) 1, 3, 6, 12, 24 hours; KCl / serum withdrawal 1, 3, 6, 12,
24 hours; control for KCl withdrawal 1, 3, 6, 12 hours; KCl
Withdrawal alone 1, 3, 6, 12 hours; control for kainic acid treatment, 2
4, 8, 12 hours; kainic acid treatment, 2, 4, 8, 12 hours; see Examples for experimental details). As shown, the genes were organized into 20 groups using 5x4 geometry. A cluster of 17 programmed cell death-inducible genes (3,3) is highlighted. The inset shows a tile representation of all genes in this (3,3) cluster (red = above average expression, white = average expression, blue = below average expression; tiles are as described above for experimental order). , Each row representing a different array element gene, in the order indicated by the distance from each cluster centroid). Caspase 3 (a gene involved in apoptosis) is part of this array and is shown in the raw value graph (ie relative expression in these 26 experiments); each experiment is represented on the x-axis in turn. The y-axis shows gene expression intensity. Figures 4B, C, D and E show the raw genes plotted for programmed cell death regulation, regulation by KCl withdrawal only, immediate early genes, and representative genes from the class of serum suppressive constitutive expression, respectively. The gene expression intensity is shown. Each panel is
Data for representative members of the cluster (indicated in this gene list by ^* ) are shown, along with a list of genes included in the expression cluster. FIG. 4B shows the raw gene expression intensities for representative genes from the list on the right. This graph shows increased expression with KCl and serum withdrawal and kainic acid treatment. Therefore, genes with these characteristics are termed "programmed cell death regulators." A list of genes with this pattern (on chip) is shown on the right. Known genes include genes that are regulated in apoptosis. FIG. 4C lists genes that show increased expression after withdrawal of KCL or KCL and serum (but after kainic acid treatment). This list includes genes known to be involved in apoptosis. FIG. 4D shows genes that show constitutive immediate early expression. FIG. 4E shows genes that show constitutive expression in the absence of serum. The list on the right indicates that this class contains mediators of programmed cell death.

FIG. 4D shows expression clusters for all CGN programmed cell death models (KCl and serum withdrawal, KCl withdrawal alone, and kainate treatment). Figure 4A
Is a self-organizing map (SOM) algorithm (eg Kohonen, Se
lf Organizing Maps: Springer, Berlin (1
997)), which was used to cluster genes based on expression in 26 experiments (Order: serum added.
ck) 1, 3, 6, 12, 24 hours; KCl / serum withdrawal 1, 3, 6, 12,
24 hours; control for KCl withdrawal 1, 3, 6, 12 hours; KCl
Withdrawal alone 1, 3, 6, 12 hours; control for kainic acid treatment, 2
4, 8, 12 hours; kainic acid treatment, 2, 4, 8, 12 hours; see Examples for experimental details). As shown, the genes were organized into 20 groups using 5x4 geometry. A cluster of 17 programmed cell death-inducible genes (3,3) is highlighted. The inset shows a tile representation of all genes in this (3,3) cluster (red = above average expression, white = average expression, blue = below average expression; tiles are as described above for experimental order). , Each row representing a different array element gene, in the order indicated by the distance from each cluster centroid). Caspase 3 (a gene involved in apoptosis) is part of this array and is shown in the raw value graph (ie relative expression in these 26 experiments); each experiment is represented on the x-axis in turn. The y-axis shows gene expression intensity. Figures 4B, C, D and E show the raw genes plotted for programmed cell death regulation, regulation by KCl withdrawal only, immediate early genes, and representative genes from the class of serum suppressive constitutive expression, respectively. The gene expression intensity is shown. Each panel is
Data for representative members of the cluster (indicated in this gene list by ^* ) are shown, along with a list of genes included in the expression cluster. FIG. 4B shows the raw gene expression intensities for representative genes from the list on the right. This graph shows increased expression with KCl and serum withdrawal and kainic acid treatment. Therefore, genes with these characteristics are termed "programmed cell death regulators." A list of genes with this pattern (on chip) is shown on the right. Known genes include genes that are regulated in apoptosis. FIG. 4C lists genes that show increased expression after withdrawal of KCL or KCL and serum (but after kainic acid treatment). This list includes genes known to be involved in apoptosis. FIG. 4D shows genes that show constitutive immediate early expression. FIG. 4E shows genes that show constitutive expression in the absence of serum. The list on the right indicates that this class contains mediators of programmed cell death.

FIG. 4 shows expression clusters for all CGN programmed cell death models (KCl and serum withdrawal, KCl withdrawal alone, and kainate treatment). Figure 4A
Is a self-organizing map (SOM) algorithm (eg Kohonen, Se
lf Organizing Maps: Springer, Berlin (1
997)), which was used to cluster genes based on expression in 26 experiments (Order: serum added.
ck) 1, 3, 6, 12, 24 hours; KCl / serum withdrawal 1, 3, 6, 12,
24 hours; control for KCl withdrawal 1, 3, 6, 12 hours; KCl
Withdrawal alone 1, 3, 6, 12 hours; control for kainic acid treatment, 2
4, 8, 12 hours; kainic acid treatment, 2, 4, 8, 12 hours; see Examples for experimental details). As shown, the genes were organized into 20 groups using 5x4 geometry. A cluster of 17 programmed cell death-inducible genes (3,3) is highlighted. The inset shows a tile representation of all genes in this (3,3) cluster (red = above average expression, white = average expression, blue = below average expression; tiles are as described above for experimental order). , Each row representing a different array element gene, in the order indicated by the distance from each cluster centroid). Caspase 3 (a gene involved in apoptosis) is part of this array and is shown in the raw value graph (ie relative expression in these 26 experiments); each experiment is represented on the x-axis in turn. The y-axis shows gene expression intensity. Figures 4B, C, D and E show the raw genes plotted for programmed cell death regulation, regulation by KCl withdrawal only, immediate early genes, and representative genes from the class of serum suppressive constitutive expression, respectively. The gene expression intensity is shown. Each panel is
Data for representative members of the cluster (indicated in this gene list by ^* ) are shown, along with a list of genes included in the expression cluster. FIG. 4B shows the raw gene expression intensities for representative genes from the list on the right. This graph shows increased expression with KCl and serum withdrawal and kainic acid treatment. Therefore, genes with these characteristics are termed "programmed cell death regulators." A list of genes with this pattern (on chip) is shown on the right. Known genes include genes that are regulated in apoptosis. FIG. 4C lists genes that show increased expression after withdrawal of KCL or KCL and serum (but after kainic acid treatment). This list includes genes known to be involved in apoptosis. FIG. 4D shows genes that show constitutive immediate early expression. FIG. 4E shows genes that show constitutive expression in the absence of serum. The list on the right indicates that this class contains mediators of programmed cell death.

FIG. 5 shows information associated with various NARC genes. Therefore, the first column is N
The ARC (neuronal apoptosis regulatory candidate) name is given. The second column provides specific information such as: number of nucleotides sequenced, sequenced region (eg 3'untranslated region), information about open reading frame, human ortholog (whose sequence is Information that may be found in SEQ ID NOs: 1-6, 8 and 10), information regarding homology to known amino acid or nucleotide sequences, information regarding function, and information relating to a particular physical or functional property. . The third column shows the gene expression class as described and presented in FIG. Column 4 shows the results of Northern blot hybridizations, such as whether expression is restricted to specific organs or ubiquitous, and transcript size.

FIG. 6 shows a table of expression data for known genes associated with programmed cell death. These data were obtained from the experiments disclosed herein where nucleic acid sequences on microarrays were hybridized to mRNA from two programmed cell death models (see Examples). . The first column shows the clone name.
If the clone is a previously known gene (eg, c-fos and c-jun), the gene name is given in addition to the cDNA clone name. Second row is BLA
The gene name of each clone based on the STX search is shown. The third column shows the expression pattern for each clone. This table can serve as an internal control for assessing the fidelity of the experimental conditions and thus as a background for comparing the expression patterns of uncharacterized clones in this array.
Therefore, this figure may serve as an internal control for discovering genes associated with apoptosis and cell proliferation.

FIG. 7 shows all genes regulated in the particular experimental conditions described in the Examples and shown in FIG. 4, ie, they are represented by the nucleic acid sequences on the chip. Cluster specific genes (underlined categories).
Each cluster represents a clone with a particular expression profile. For example, the first cluster is transiently downregulated by serum and by KCl withdrawal. The second column is the cD whose function was previously known.
Identify NA clones. The third column shows the number of clusters. See Figure 4A. Furthermore, analysis of the function of genes in each cluster indicates that within the cluster, certain functional classes of genes may be over-represented. Thus, the material in brackets indicates the biological function associated with the disproportionate number of genes in that cluster. This includes secretory and synaptic vesicle release (cluster 0,0), cell proliferation (cluster 0,3), secretion / synaptic vesicle release / cytoskeletal remodeling (cluster 1,0).
, Stress response / hormone response (cluster 1,3), stress response / hormone response (cluster 1,4), calcium signaling (cluster 2,0), and cytoskeleton / synaptic cytoskeleton (cluster 2,4). To be

FIG. 8 summarizes tissue expression data for Smart Chip I ^™ microarray elements. These data were obtained by microarray membrane blotting of mRNA from testis, brain, heart, smooth muscle, spleen, kidney, skeletal muscle, lung, liver and pancreatic tissues. After hybridization with labeled cDNA synthesized from RNAs from the indicated tissue types, the signal from each sequence on the array filter was quantified by fluorescence imaging.

FIG. 9 provides a list of genes shown to be regulated by KCl and serum withdrawal in the microarray experiments described herein.

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Claims (28)

[Claims] 1. An isolated nucleic acid molecule, which comprises: (a) the nucleotide sequences set forth in SEQ ID NOs: 1-6, 8 and 10, and (b) SEQ ID NOs: 1-6. , The complement of the nucleotide sequences shown in 8 and 10,
A nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: 2. An isolated nucleic acid molecule, the nucleic acid molecule comprising: a) the nucleotide sequences set forth in SEQ ID NOs: 1-6, 8 and 10, and b) the complement of the nucleotide sequences shown in SEQ ID NOs: 1-6, 8 and 10. A nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of: 3. An isolated nucleic acid molecule, the nucleic acid molecule comprising: a) the nucleotide sequences set forth in SEQ ID NOs: 1-6, 8 and 10, and b) SEQ ID NOs: 1-6, 8. , And a complement of the nucleotide sequence set forth in 10, wherein the nucleic acid molecule is a fragment of a nucleotide sequence selected from the group consisting of, wherein the fragment is at least 15 nucleotides in length. 4. A group consisting of: a) the nucleotide sequences shown in SEQ ID NOS: 1-6, 8 and 10, and b) the complement of the nucleotide sequences shown in SEQ ID NOS: 1-6, 8 and 10. A nucleic acid molecule comprising a nucleotide sequence that is at least 60% identical to a nucleotide sequence selected from. 5. A group consisting of: a) the nucleotide sequences shown in SEQ ID NOS: 1-6, 8 and 10, and b) the complement of the nucleotide sequences shown in SEQ ID NOS: 1-6, 8 and 10. A nucleic acid molecule that hybridizes under high stringency conditions to a nucleotide sequence selected from. 6. The following: a) the nucleotide sequences set forth in SEQ ID NOs: 1-6, 8 and 10, b) the nucleotides that are at least 60% identical to the nucleotide sequences set forth in SEQ ID NOs: 1-6, 8 and 10. A sequence, c) a nucleotide sequence that hybridizes to the nucleotide sequences shown in SEQ ID NOs: 1 to 6, 8 and 10 under high stringency conditions, and d) a complement of the nucleotide sequence of a, b or c. A vector comprising a nucleotide sequence selected from the group. 7. The vector of claim 6, wherein the isolated nucleic acid molecule is operably linked to at least one expression control element. 8. A host cell containing the vector of claim 7. 9. A method for preparing a polypeptide comprising culturing a host cell according to claim 8 under conditions in which the nucleic acid molecule is expressed. 10. An isolated polypeptide encoded by the nucleic acid molecule of claim 1. 11. An isolated polypeptide encoded by the nucleic acid molecule of claim 4. 12. An isolated polypeptide encoded by the nucleic acid molecule of claim 5. 13. An antibody that selectively binds to the polypeptide of claim 10. 14. An antibody that selectively binds to the polypeptide of claim 11. 15. An antibody that selectively binds to the polypeptide of claim 12. 16. A method for assaying for the presence of a nucleic acid molecule in a sample, the method comprising the steps of: (a) contacting the sample with a nucleic acid probe that selectively hybridizes to the nucleic acid molecule. The nucleotide sequence shown in SEQ ID NOs: 1-6, 8 and 10; the complement of the sequence shown in SEQ ID NOs: 1-6, 8 and 10; at least 15 nucleotides in length Fragments of the nucleotide sequences set forth in SEQ ID NOs: 1-6, 8 and 10; and at least 1
Selected from fragments of the complement of the nucleotide sequences set forth in SEQ ID NOS: 1-6, 8 and 10, which are 5 nucleotides; and (b) whether the nucleic acid probe binds to a nucleic acid molecule in a nuclear sample. Deciding whether or not. 17. A method for detecting the polypeptide of claim 10 in a sample, the method comprising the steps of: (a) an antibody that binds to the polypeptide of claim 10. Contacting the sample, and (b) determining whether the compound binds to the polypeptide in the sample. 18. A method for modulating the activity of the polypeptide of claim 10, wherein the method allows the agent to modulate the activity of the polypeptide. 11. A method comprising contacting the polypeptide according to 10 with the factor. 19. The method of claim 18, wherein the factor is an antibody that binds to the polypeptide. 20. The method of claim 18, wherein the polypeptide is present in cells derived from the central nervous system. 21. The method of claim 18, wherein the cells derived from the central nervous system undergo aberrant apoptosis. 22. The method of claim 18, wherein the activity is modulated in a subject having or susceptible to a disorder associated with the central nervous system. 23. The method of claim 18, wherein the activity is modulated in a patient having or susceptible to a disorder associated with aberrant apoptosis. 24. A method for treating a central nervous system-related disorder, comprising:
11. A method comprising the step of administering to a patient having or at risk of developing the disorder any of the polypeptides of claim 10. 25. A method for treating a disorder associated with aberrant apoptosis, the method comprising subjecting or at risk of developing the disorder,
A method comprising the step of administering any of the polypeptides of claim 10. 26. A kit comprising a nucleic acid probe that hybridizes to the nucleotide sequence of claim 1 and instructions for use. 27. A kit comprising an agent that binds to the polypeptide of claim 10 and instructions for use. 28. The kit of claim 35, wherein the factor is an antibody.