WO2001030973A2 - Genetic sequences associated with neural cell proliferation and disease - Google Patents

Abstract

Applying large-scale DNA microarray techniques, the developmental molecular programs of the hippocampus, a cortical structure critical for learning and memory has been anlyzed. Over 30,000 genes and EST sequences were screened, including an estimated 30-40% of total genes in the mouse genome. As a result, the present invention identifies 4,390 genes that showed dynamic changes during hippocampal development. Moreover, using genecluster analysis, these genes were grouped into 16 distinct clusters with striking patterns that correlate with major developmental hallmarks. The covariance analysis strategy was then applied. This enabled the identification of a group of genes whose expression may underlie the phenotypic changes in the hippocampus.

Description

GENETIC SEQUENCES ASSOCIATED WITH NEURAL CELL PROLIFERATION AND DISEASE

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Nos. 60/161,337, filed October 25, 1999, and 60/227,639, filed August 24, 2000, the entireties of which are incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to the fields of molecular biology and neuronal growth and development. Specifically, the invention provides a method of screening large numbers of genes and analyzing expression patterns of genes. The invention also provides nucleic acid sequences which may be used to advantage for designing therapeutic agents useful for the treatment of cancer, neuronal degeneration diseases, nerve injury and aging.

BACKGROUND

DNA mircroarray technology offers the genome-wide view of molecular and genetic events of biological processes: simultaneous readouts of all aspects of transcriptional cascades. Micro fabricated arrays of large numbers of oligonucleotide probes offer great promise for a wide variety of applications, including the ability to monitor large-scale gene expression during development, as well as during different functional or dysfunctional stages in adult organisms. GeneChip® probe arrays (Affymetrix, Santa Clara, CA) are high-density oligonucleotide microarrays that enable simultaneous hybridization-based analysis of thousands of genes. Using Genechip analysis, it is possible to scan significant portions of the genome in order to profile comprehensive molecular and genetic programs underlying various aspects of development.

The complex and diverse functions of the mature brain depend on the precise interconnections formed by thousands of neural cell types. The mammalian brain develops in a series of ordered steps, with a precise temporal sequence that is characteristic of each subregion. The hippocampus is a brain structure that plays a critical role in learning and memory in both humans and other animals. It is generally believed that various processes such as proliferation, differentiation, synapse formation, maturation of synaptic function are achieved by the activation of specific sets of genes within the cells controlled by genetic programs which can also be modified by environments. While molecular and cellular mechanisms underlying hippocampal synaptic plasticity have been studied extensively, the genetic programs underlying the development of the hippocampus remain largely undefined. Thus, there is a need in the art for an enhanced understanding of the hippocampus and its role in various physiological and pathological conditions.

SUMMARY OF THE INVENTION

The present invention is based on the discovery of genes whose expression is linked to distinct cellular phenomena such as cell division and proliferation during the development of the brain.

It is another object of the present invention to provide a method for isolating protein or protein coding sequences that correspond to any one of the sequences set forth in Tables 1 or 2 comprising the steps of assembling a hybridization reaction mixture containing one or more of the isolated nucleic acid molecules in single stranded form, and a test sample that comprises the corresponding protein coding sequence in a single-stranded form, under conditions enabling hybridization of the isolated nucleic acid molecule and the protein sequence, thereby forming a double-stranded nucleic acid molecule; separating the double-stranded molecule comprising the isolated nucleic acid and the protein coding sequence; and optionally cloning the protein coding sequence. According to an additional aspect of the invention, isolated proteins produced by expression of the aforementioned protein coding sequences are provided. Also provided in the present invention are antibodies immunologically specific for the protein molecules.

It is another object of the invention to provide a method of screening and analyzing the expression patterns of genes in various developmental stages of the hippocampus. The method comprises hybridizing isolated brain mRNA to an oligonucleotide array, clustering groups of genes together using self- organizing map analysis, and analyzing alterations of expression levels of genes. In one preferred embodiments, the mRNA is hippocampal mRNA. In a second preferred embodiment, the mRNA is comprised of polynucleotide sequences of Tables 1 or 2. In other preferred embodiments, the developmental stages are selected from the group consisting of embryonic day 16, postnatal day 1 , postnatal day 7, postnatal day 16, and postnatal day 30.

The present invention also provides nucleic acid sequences associated with neural development and cell proliferation. These sequences are provided with their accession numbers in Tables 1 and 2 and are expressly incorporated herein by reference. The proteins encoded by these nucleic acid molecules provide novel biological targets for neuronal disorders associated with the aberrant expression of the brain development related nucleic acids of the invention.

Also provided for in the present invention is a method of screening for therapeutic agents which induce or inhibit expression of genes in the hippocampus comprising the steps of contacting a hippocampal cell with a test substance; and assaying for the amount of expression in the cell of two or more genes of Table 1 and/or Table 2, wherein the expression of said genes in the cell is assayed before and after the cell has been contacted with the test substance, and wherein the therapeutic agent is identified if it increases or decreases expression of at least one of said genes.

Also provided for in the present invention are methods of treating disease including administering to a diseased patient a therapeutically effective amount of a gene from Table 1, or a polynucleotide sequence selected from Table 2. Other therapeutic methods provided for include administering to a diseased patient a therapeutically effective amount of a polypeptide that competes with the polypeptide encoded by any one of the genes or their corresponding nucleotide sequences set forth in Table 1 or Table 2 for the polypeptide's ligand, substrate, or receptor.

In a further aspect of the invention, the sequences provided are utilized as primers to amplify the full-length corresponding nucleic acids. Vectors comprising the sequences of the invention as well as host cells containing the same are contemplated to be within the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a dissection time line of the developing mouse hippocampus. The arrows illustrate a few examples of the peak windows for the major neuronal events occurred during the hippocampal development.

Fig. 2a shows reproducibility of genes identified by microarray technology and obtained from GeneChip7k array study. The green dots represent the genes detected in both PI samples, an estimated 34% of the genes on a single chip. The yellow or blue dots represent the genes detected only in one of the sample, and thus these signals only constitute about 0.25- 0.3% of the genes on the chip.

Fig. 2b depicts differential gene expression study between PI and P30. The axes are intensities of genes. An intensity of 400 to 500 corresponds to approximately one copy per cell. The axes represent gene expression intensity. The green dots represent the genes detected in both PI and P30. The yellow dots represent the genes detected in PI, but not P30. Similarly, the blue dots represent the genes detected in P30 sample but not in PI. 5% of probe sets showed more than 3 fold changes.

Fig. 3a this figure is a self-organizing map (SOM) were used to group the 4,390 identified genes and ESTs into clusters based on similar expression dynamics over the five time point represents cluster analysis of probes sets on mouse 1 IK arrays.

Fig. 3b this figure is a self-organizing map (SOM) were used to group the 4,390 identified genes and ESTs into clusters based on similar expression dynamics over the five time point represents cluster analysis of probe sets on 19K arrays. Analysis algorithms were used to convert raw data into expression data for these genes before applying SOM analysis. The label at the upper-left corner of each inset represents the appropriate cluster number (from C0-C15). The number in the top center of each inset represents the number of genes in that particular cluster. The five dots in each inset represent the five developmental time points (El 6, PI, P7, PI 6, and P30).

Fig. 4 shows the biochemical pathway of glycolysis. The genes identified from the pathway are shown as marked in yellow.

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to the first large-scale gene expression analysis in the brain, and is aimed to link the genomic expression patterns with distinct cellular phenotypes. The inventive expression analysis identified a large number of genes whose expression is associated with neuronal cell division, proliferation and differentiation. The present invention is also the first genome-wide search for genes that control cell cycles and differentiation in the brain.

It is known that the most active proliferation of neurons in mice occurs during the prenatal period, followed by dynamic outgrowth and differentiation around the neonatal period before the synaptogenesis in the first postnatal week. By the second postnatal week, synaptic connections are established and synaptic activity becomes increasingly active. By the third postnatal week, the fully connected hippocampal circuits exhibit great plasticity such as robust long-term potentiation. By the end of the first month, the hippocampus has entered into a more mature state (Jacobson, M., Developmental Neurobiology. 3^rd ed. 1991, New York: Plenum Press, ix, 776; Isaacson, R.L., et al., The Hippocampus. New York: Plenum Press; Pokorny, J., et al., (1981) Brain Res. Bull. 7, 113-120; Pokorny, J., et al., (1981) Brain Res. Bull. 7, 121-130).

To analyze the genome-wide molecular events that occur in the developing hippocampus, the present invention employes the GeneChip7k technology. GeneChip7k arrays are high-density oligonucleotide microarrays, enabling hybridization-based analysis of thousands of genes simultaneously (Lipshultz, et. al., (1999) Nature genetics 21, supplement, 20-24). The current Affymetrix mouse arrays contain approximately 11,000 known genes and 19,000 EST sequences, or approximately 30-40% of total mouse genes.

Therefore, GeneChip7k permits scanning significant portions of the genome and profiling of comprehensive molecular and genetic programs underlying the mammalian brain development. To establish comprehensive gene expression profiles, five time points were chosen between the embryonic day 16 to postnatal day 30. The five time points, embryonic day 16 (El 6), postnatal day 1 (PI), postnatal day 7 (P7), postnatal day 16 (PI 6) and postnatal day 30 (P30) correspond to the peak times during which major cellular and physiological events occur during mouse hippocampal development, as shown in Figure 1.

In the present invention, hundreds of hippocampi were dissected from developing mouse brains at each of the five time points and separated into two independent tissue pools for extraction of poly(A) mRNA. These duplicate mRNAs were used to generate fluorescently labeled targets for hybridization to microarrays containing over 30,000 mouse genes and EST sequences. Each array comprises approximately 6,500 genes or EST sequences. In order to ensure reliability of data, all hybridization of the present invention was conducted in duplicate, namely two independent mRNAs and two sets of duplicate microarrays were used.

When expression levels of the same set of genes were compared between two developmental time points, such as PI versus P30, it was found that 325 genes on the chip were differentially expressed with more than threefold changes, as shown in Figure 2b. Out of over 33,945 probe sets screened, over 4,390 probe sets exhibited dynamic changes during the hippocampal development period between El 6 to P30.

To explore the patterns and possible genetic principles underlying the development-regulated gene expression, a powerful cluster analysis tool was employed, namely self-organizing map analysis (SOM) (Tamayo, P., et al., (1999) PNAS USA 96, 2907-2912). This mathematical cluster analysis enabled the recognition of expression patterns that were not detectable through the conventional gene-by-gene approach. As a result, the SOM approach permits the classification of genes in a manner that is completely independent of their sequence information.

The first genes analyzed for the present invention were the 1 ,926 known genes that showed significant expression changes during development. These 1926 genes were grouped into sixteen distinct gene clusters, as shown in Table 1 and Figure 3 a. Generally, these sixteen clusters can be further classified into four major types, Type I, Type π, Type HI and Type IV, each comprising one or more clusters of genes (C0-C15). These clusters correlate with major cellular changes during the hippocampal development.

Type I includes the clusters CO, CI and C5 and exhibits an overall age- dependent down-regulation. In Type I, a total of 228 genes exhibited high expression at E16, but essentially switched off after birth. This swift genetic switch is correlated with the well-known phenotypic changes and transition of neurons from the proliferating to post-mitotic state. Surprisingly, most of these 228 genes are known to be involved in the control of cell cycles, histone regulation, and DNA replication.

Type II comprises CIO, C11, C14 and C15 and shows a general age- dependent increased expression which reaches peak levels either at PI 6 or P30.

Type III comprises the clusters C4, C8, C12, C13 and C14 and shows peak expression at either PI or P7. Cluster C12 in Type in shows a dramatic up-regulation of expression at postnatal day 1 with low expression at other time points. This cluster contains genes known to be involved in cell-type specification, such as Nkx, homeobox transcription factors (Pabst, O., (2000) Dev. Genes Evol. 210, 47-50), morphogenetic events (including Wnt-3 and Wnt-lOa), and post-mitotic regulation of neuronal differentiation, including the zinc finger family gene Postmitotic Neural Gene-1 ("Png-1") (Weiner, JA, et al., (1997) J. Comp. Neurol. 381, 130-142). Therefore, the cluster method of the present invention appears to accurately reflect the potential underlying molecular and genetic programs during hippocampal development.

Type IV includes the clusters C2, C3 and C6, and exhibits down- regulation at PI or P7.

In addition to analyzing the genes known to show significant expression changes during hippocampal development, the inventive method was also conducted on 2,464 EST sequences, resulting in the establishment of several clusters as shown in Figure 3b and the identification of 349 novel polynucleotide sequences set forth in Table 2. Thus, the clusters of the present invention are useful in assigning cellular phenotypes to genes within the cluster, and thereby facilitating the functional discovery processes of these previously unidentified genes. Many of the novel genes of the present invention were found to have expression that varied greatly between different developmental stages and between different organs in the same developmental stages. Thus, these genes are useful as interrogators (probes) to determine the expression pattern of unknown cells or samples to identify a sample of tissue or cell as belonging to the appropriate developmental stage or organ source. The genes of the present invention can also be used to identify human homologues and as tools in the development of therapeutic drugs for the treatment of neuronal degeneration diseases, nerve injuries, aging, and cancer.

In one embodiment, A method of screening for candidate drugs which induce or inhibit expression of genes in the hippocampus comprises the steps of contacting a hippocampal cell with a candidate drug for sufficient time for detectable expression of a gene and assaying for the amount of expression in the cell of two or more genes selected from the group consisting of Tables 1 and 2 wherein the expression of these genes in the cell is assayed before and after the cell has been contacted with the test substance. The candidate drug is then identified if it increases or decreases expression of one or more of these genes.

Any selection of at least two genes can be used as interrogators for purposes of the present invention. For a particular interrogation of two conditions or sources, it is desirable to select those genes which display a great deal of difference in the expression pattern between the two conditions or sources. At least a two-fold difference is desirable, but a three-fold, five-fold or ten-fold difference is preferred.

Also provided for in the present invention is a method of treating a disease comprising administering to a diseased patient a therapeutically effective amount of a gene from Table 1 or Table 2 or by administering to a diseased patient a therapeutically effective amount of a polypeptide that competes with the polypeptide encoded by any one of the genes in Table 1 or Table 2 for its ligand, substrate, or receptor. A pharmaceutically acceptable carrier or excipient may optionally be included with the therapeutically effective amount of the gene. Such carriers include but are not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. Formulation should suit the mode of administration, and is well within the skill of the art. Suitable forms of administration include intravenous, subcutaneous, intramuscular, or intraperitoneal injection. Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents. In addition, if properly formulated in enteric or encapsulated formulations, oral administration may also be possible.

Interrogators of the genes or proteins can be performed to yield different information. Potential drugs can be screened to determine if the expression of these genes is inappropriately altered. This is useful for example, in determining whether a particular drug is prescribed to a class or subclass of individuals. In the case where a fetal gene's expression is affected by the potential drug, prohibition of the drug to pregnant women is indicated. Similarly, a drug which causes expression of a gene which is not normally expressed by a fetus should be prohibited to pregnant women.

Molecular expression markers for brain tissue, and in preferred embodiments, hippocampal tissue, can be used to confirm tissue source identifications made on the basis of morpho logical criteria. In some situations, identifications of cell type or source is ambiguous based on classical criteria but can be easily identifiable using the methods of the present invention. In these situations, the molecular expression markers of the present invention as described in Tables 1 and 2 are useful.

In addition, disease progression involving brain tissue can be monitored by following the expression patterns of the affected brain tissue using the molecular expression markers of the present invention. Altered expression can be observed in the diseased state. Monitoring of the efficacy of certain drug regimens can also be accomplished by following the expression patterns of the molecular expression markers set forth in Tables 1 and 2.

Neuronal disorders can be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of genes of the present invention or their corresponding mRNA. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods. Assay techniques that can be used to determine levels of a protein in a sample derived from a host are well- known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays. The genes which are assayed or interrogated according to the present invention are typically in the form of mRNA or reverse transcribed mRNA. The genes may be cloned and/or amplified. The cloning itself does not appear to bias the representation of genes within a population. However, it may be preferable to use polyA+ RNA as a source, as it can be used with less processing steps. The sequences of the expression marker genes of Table 1 and Table 2 are in the public databases. The sequences of the genes listed in Table 1 and Table 2 are expressly incorporated herein by reference. Some of the genes are also the subject of scientific and journal articles as described below. Each article referenced below is also expressly incorporated by reference herein.

Fragmented oligonucleotide probes for interrogating the tissue or cell sample are preferably of sufficient length to specifically hybridize only to appropriate complementary genes or transcripts. Typically the oligonucleotide probes will be at least 10, 12, 14, 16, 18, 20 or 25 nucleotides in length. In some cases, longer probes of at least 30, 40 or 50 nucleotides are desirable.

In preferred embodiments of the present invention, solid phase arrays are used for the rapid and specific detection of nucleic acid molecules and their expression patterns. Typically, a probe is linked to a solid support and a target nucleic acid (e.g., a genomic nucleic acid, an amplicon, or, most commonly, an amplified mixture) is hybridized to the probe. Either the probe, or the target, or both, can be labeled, typically with a fluorophore or other tag, such as streptavidin. Where the target is labeled, hybridization is detected by detecting bound fluorescence. Where the probe is labeled, hybridization is typically detected by quenching of the label. Where both the probe and the target are labeled, detection of hybridization is typically performed by monitoring a color shift resulting from proximity of the two bound labels. A variety of labeling strategies, labels, and the like, particularly for fluorescent based applications are described, supra.

Probes of the present invention are synthesized on a solid support. Exemplary solid supports include glass, plastics, polymers, metals, metalloids, ceramics, organics, etc. Using chip masking technologies and photoprotecfive chemistry it is possible to generate ordered arrays of nucleic acid probes. These arrays, which are known, e.g., as "DNA chips," or as very large scale immobilized polymer arrays ("VLSEPS.TM." arrays) can include millions of defined probe regions on a substrate having an area of about 1 cm² to several cm², thereby incorporating sets of from a few to millions of probes.

The construction and use of solid phase nucleic acid arrays to detect target nucleic acids is well described in the literature. See, Fodor et al. (1991) Science, 251 : 767-777; Sheldon et al. (1993) Clinical Chemistry 39(4): 718- 719; Kozal et al. (1996) Nature Medicine 2(7): 753-759 and Hubbell U.S. Pat. No. 5,571,639. See also, Pinkel et al. PCT/US95/16155 (WO 96/17958). Typically, a combinatorial strategy allows for the synthesis of arrays containing a large number of probes using a minimal number of synthetic steps. For instance, it is possible to synthesize and attach all possible DNA 8- mer oligonucleotides (48, or 65,536 possible combinations) using only 32 chemical synthetic steps.

Light-directed combinatorial synthesis of oligonucleotide arrays on a glass surface is performed with automated phosphoramidite chemistry and chip masking techniques similar to photoresist technologies in the computer chip industry. Typically, a glass surface is derivatized with a silane reagent containing a functional group, e.g., a hydroxyl or amine group blocked by a photolabile protecting group. Photolysis through a photolithogaphic mask is used selectively to expose functional groups which are then ready to react with incoming 5'-photoprotected nucleoside phosphoramidites. The phosphoramidites react only with those sites which are illuminated (and thus exposed by removal of the photolabile blocking group). Thus, the phosphoramidites only add to those areas selectively exposed from the preceding step. These steps are repeated until the desired array of sequences have been synthesized on the solid surface. Combinatorial synthesis of different oligonucleotide analogues at different locations on the array is determined by the pattern of illumination during synthesis and the order of addition of coupling reagents. Monitoring of hybridization of target nucleic acids to the array is typically performed with fluorescence microscopes or laser scanning microscopes.

In addition to being able to design, build and use probe arrays using available techniques, one of skill is also able to order custom-made arrays and array-reading devices from manufacturers specializing in array manufacture. For example, Affymetrix Corp., in Santa Clara, Calif, manufactures DNA VLSIP.TM. arrays.

Genetic Events During the Prenatal Period

The profiling and gene cluster analysis of the present invention permits the evaluation of overall molecular patterns on a genome-wide scale. This approach also allows gene activities to be assigned with phenotypic cellular events. For example, from E16 to PI, almost all of the hippocampal neurons switch from a highly active proliferation state to a post-mitotic state. As such, expression of proliferative genes involved in cell cycle progression are highly expressed at El 6, and then subsequently become silent or reduced significantly after birth. These genes were prominently grouped in Cluster 1 as shown in Table 1. Cluster 1 genes appear to correspond to general cellular features that are peaked in early development, including proliferation, DNA and RNA synthesis, and transcriptional and translational regulation.

The cyclin family of proteins that regulate mitosis are represented in Cluster 1 by cyclin-dependent kinase regulatory subunit-2, cyclins B2 and G2, D-type cyclin, Gl/S-specific cyclin D2, D-type Gl cyclin catalytic subunit, and cell division protein kinase 4 (Endicott, JA, et al., (1999) Curr. Opin. Struct. Biol. 9, 738-74; Home, MC, et al., (1996) J. Biol. Chem., 271, 6050- 6061; Sherr, CJ (1995) Trends Biochem. Sci. 20, 187-90). GADD45, a growth arrest protein (Sheikh, MS, et al., (2000) Biochem. Pharmacol. 59, 43-45) was also identified as being in Cluster 1, suggesting a balance of factors that regulate mitosis.

Another group of genes whose expression are believed to contribute to the high proliferative activity of embryonic brain are those that encode enzymes essential for DNA and RNA synthesis. These are also found in Cluster 1. For example, DNA topoisomerase II (Berger, JM (1998) Curr. Opin. Struct. Biol. 8, 26-32) was identified in this cluster. This enzyme makes double-stranded breaks to control the topological state of DNA during replication. Many of these factors are involved in RNA processing and include RNA polymerase I, an enzyme which catalyzes the transcription of DNA to RNA. Also included in this group is the DEAD family of RNA helicases that regulate ribosome assembly, pre-mRNA splicing, mRNA translation and RNA stability by using intrinsic ATPase activity to catalyze conformational changes in RNA secondary structure (Luking A., et al., (1998) Crit. Rev. Biochem. Mol. Biol. 33, 59-96). Other pre-mRNA splicing factors were identified in Cluster 1, including unwinding protein 1 (Jiang J, et al., (2000) PNAS USA 97, 3022-3027), U2 and U6 snRNPs (Ro-Choi TS (1999) Crit. Rev. Eukaryot. Gene Expr. 9, 107-158), SRP75 from the SR family, as well as the myoblast cell surface antigen 24 which has been identified as a pre-mRNA splicing factor (Gower HJ, et al., (1989) Development. 105, 723-731). In addition, chromosomal proteins H2A.X, H2A.1 and HI histone subtype H1(0) (Doenecke, D, et al. (1997) Histochem. Cell Biol. 107, 1-10; Doenecke D, et al. (1997) J. Cell Biochem. 54, 423-31) were also identified in cluster 1. Interestingly, several key transcription factors were identified in cluster 1. These include BTF3, a transcription factor required for transcription initiation of RNA polymerase II (Grein S, et. al. (1999) Mol. Cell Biochem. 191, 121-128) and NF1-B, which mediates the transcription of several differentiation markers (Osada S, et al. (1999) Biochem J. 342, 189-198). Neurogenin-2, which encodes a neural-specific basic helix-loop-helix protein that is involved in determination of neuronal fate, specification and differentiation of neuronal cell lineages (Ma Q, et al., (1999) Genes Dev. 13, 1717-1728) was also identified in Cluster 1.

In addition to the transcriptional factors, many genes known to be involved in translational regulation were also identified in Cluster 1. These genes include the initiation factor 2, elongation factor- 1 and B2 (Clark BF, et al. (1999) FEBS Lett. 452, 41-46). Elongation factor-1 is a G-protein that mediates the transport of aminoacyl tRNA to 80S ribosomes during translation. Elongation factor-2 is a protein that promotes the GTP-dependent translocation of the nascent protein from the A-site to the P-site of the protein. Many types of ribosomal proteins were also up regulated at this time (see Table 1). Profiling analysis indicates that the high rate of protein synthesis of protein in embryonic hippocampus is also coupled with the up-regulation of genes involved in protein degradation, suggesting that it is a tightly controlled process. For example, ubiquitin-conjugating enzyme E2 (Yamao F. (1999) J. Biochem (Tokyo) 25, 223-229), involved in ubiquitin-mediated protein degradation was also identified, suggesting a regulation of protein turnover at E16.

Genetic Events During The Neonatal And Early Postnatal Development

The hippocampus undergoes phenotypic changes after birth. The overwhelming majority of the neurons enters the post-mitotic state and show extensive growth and differentiation in the first postnatal week. These cellular changes are marked by rapid cytoskeletal changes, production of cell adhesion molecules, and extracellular matrix formation, as well as expansion of cell membrane.

Appropriately, the genes whose activities are highly active during this time are mostly represented by clusters 4 and 8 as shown in Table 1. These two clusters have similar yet distinct profiles with a peak occurring at P 1 that decreased to basal levels by PI 6. Many actin and tubulin isoforms were identified in these clusters, including beta- and gamma-actin, actin- 1 and actin- 3 , as well as several forms of alpha- and beta-tubulin, consisting with the notion that cellular differentiation is associated with dynamic production of cytoskeletal and structural proteins (Bradke F., et al. (2000) Microsc. Res. Tech. 48, 3-11). Genes encoding the CCT chaperonin containing family are prominently expressed in the same cluster. These chaperonin proteins are essential for promoting the correct folding of actin and tubulin (Lund PA. (1995) Essays. Biochem. 29, 113-123). We observed that the subunits beta, epsilon, delta, and theta of the CCT chaperonin family are all highly expressed in the neonatal and early postnatal days.

Also up-regulated during the neonatal and early postnatal period are several cell adhesion and extracellular matrix proteins. These proteins are known to play critical roles during the differentiation and pattern formation. They include collagen and fibronectin, LI -like protein (Hillenbrand R. et al. (1999) Eur. J. Neurosci. 11, 813-826), a neural cell adhesion molecule, and neural cell adhesion molecule LI (Baldwin TJ, et al. (1996) J. Cell. Biochem. 61, 502-513), neurophilin and neural cadherin (Jessell TM, et al. (1990) Annu. Rev. Neurosci. 13, 227-255).

Morphological differentiation is typically associated with membrane expansion. Several genes were found to be involved in fatty acid and membrane synthesis are up-regulated in the neonatal and early postnatal period. They include brain fatty acid binding protein (B-FABP), fatty acid binding protein (FABP) and fatty acid synthase. These molecules are the building blocks of phospholipids and glycolipids (Glatz JF, et al. (1996) Prog. Lipid Res. 35, 243-282), which are important components of the differentiation that is occurring at the PI neonatal time period.

Genetic Switches Up-Regulated In The Late Postnatal Hippocampus

Following the neuronal differentiation and synapse formation in the early postnatal week, the hippocampal synapses and circuits become more active and begin to exhibit more exuberant plasticity. Several clusters show a gradual increase in expression and are up-regulated at either PI 6 or P30. These genes were typically represented in clusters 11 and 15 can be organized into categories of synaptic function, signal transduction, transcriptional and translational control, glucose and oxidative metabolism and membrane regulation of ionic concentration.

For example, many genes in clusters 11 and 15 are involved in synaptic function. Some of these are involved in synaptic vesicle trafficking, including clathrin, which is a component of the coat that surrounds vesicles and synaptogamin, a vesicle-associated protein involved in calcium-mediated release of neurotransmitter. Two synaptic vesicle-associated proteins, VAMP2 and synaptophysin were identified (Bennett MK, et al. (1992) J. Cell Biol. 116, 761-775; Calakos, N et al., (1996) Physiol. Rev. 76, 1-29), as well as UNC-18, which is believed to play a role in vesicle trafficking (Hata Y, et al. (1993) Nature 366, 347-351).

In addition to the genes involved in vesicular trafficking, several presynaptically released neuromodulators were also identified. These include the chemokine fractalkine (Meucci O. et al. (1998) PNAS USA 95, 14500- 14505), cholecystokinin (Kelly JS et al (1981) Adv. Biochem.

Psychopharmacol. 28, 133-144) and brain-derived neurotrophic factor (BDNF), a neurotrophin. Furthermore, many neurotransmitter receptors were identified, which would play a role in the synaptic function on the postsynaptic side. These include neurotransmitter receptors for glutamate, including glutamate receptor 1 (GluRl), glutamate receptor 2 (GluR2) and the NMD A receptor, as well as a receptor for acetylcholine (AP). In addition, the neurotensin receptor was identified (Vincent JP, et al. (1999) Trends Pharmacol. Sci. 20, 302-309).

In association with maintaining membrane potential, several molecules that help to maintain ionic concentrations across membrane were identified as up-regulated in the late postnatal period. They include potassium channels, vacuolar adenosine triphosphatase (pore forming subunits B and E) and transporters that catalyze sodium and potassium co-transport.

To ensure the correct signal transduction between neurons, the proper intracellular signal transduction must be established. Indeed, we found that many signaling molecules are coordinated with synaptic activity and synaptic plasticity at the postnatal developmental stages. For example, receptor type tyrosine kinase was identified as well as many of the proteins that are involved in tyrosine kinase signaling pathways, such as ras, ras-related protein RAB-3A, and mito gen-activated protein kinase (erk-1). In addition, calcineurin B, a protein phosphatase regulatory subunit, and FK506-binding protein (also known as PKBP-12) which bind to and inhibit calcineurin, are also present in this cluster. These proteins have been shown to be involved in regulating synaptic plasticity (Mulkey RM, et al. (1994) Nature 369, 486-488).

A set of transcriptional and translational factors was identified whose expression exhibit the gradual increase during the post-natal weeks. These transcriptional and translational factors are different from the transcriptional and translation factors found in the embryonic hippocampus. No overlap of factors identified in cluster 1 vs. clusters 11 and 15 was observed, suggesting that these temporally regulated transcription events are distinct and likely control different developmental phenotypes. For example, zif7268 (Vincent JP et al. (1999) Trends Pharmacol. Sci. 20, 302-309), DNA binding protein SMBP2 (Cox GA et al. (1998) Neuron. 21, 1327-1337), transcriptional activator FE65 (Faraonio R. et al. (1994) Nucleic Acids Res. 22, 4876-4883) and transcription factor Sox-M were identified.

In addition to changes in synaptic properties, it is well known that the high level of synaptic activity needs to be supported by efficient energy utilization and production. In fact, the brain's energy metabolism switches from ketone in neonatal brain to glucose in the adult brain as a major energy fuels. Surprisingly, we have found that many of the enzymes involved in glucose metabolism and oxidative metabolism are gradually up-regulated as a function of postnatal development. A complete list is provided in Table 1. In Type II clusters (primarily in the CI 5 of 1 IK SOM), all the major enzymes involved in glycolysis were identified by gene-cluster analysis. This observation not only validates the approach, but also fits nicely with the well- known fact that the energy utilization of the mammalian brain switches from ketone in neonatal stages to glucose at more mature stage. As shown in Figure 4, the glycolysis pathway converts glucose into pyruvate, generating 2 molecules of ATP per reaction. The identification of all the key glycolytic enzymes in clusters 11 and 15 supports this transition. These genes include 2 key enzymes that control the pace of glycolysis, phosphofructokinase and pyruvate kinase. Other genes include glucose-6-phosphate isomerase, fructose bisphosphate aldolase A and C, triose phosphate isomerase, phosphoglycerate kinase and neural enolase (Tani M. et al. (1997) Genomics 39, 30-37).

The oxidative metabolic pathway was also significantly up regulated with age. Some of the molecules involved in the mitochondrial respiratory chain include NADH-ubiquinone oxidoreductase, cytochrome c oxidase, succinate dehydrogenase, malate dehydrogenase, lactate dehydrogenase and glycerophosphate dehydrogenase (Stryer L. Biochemistry, Freeman, New York, 1995).

Therefore, these postnatal onset genes are primarily involved in regulation of synaptic function, signal transduction, transcriptional and translational control, glucose and oxidative metabolism as well as membrane regulation of ionic concentration.

Definitions

Various terms relating to the biological molecules of the present invention are used throughout the specification and claims.

"Antibodies" as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library.

"Isolated" means altered "by the hand of man" from the natural state. If an "isolated" composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not

"isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein.

"Polynucleotide" generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides" include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications has been made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.

"Polypeptide" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. "Polypeptides" include amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.

Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that 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 many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross- linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993 and Wold, F.,

Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al., "Analysis for protein modifications and nonprotein co factors", Meth Enzymol (1990) 182:626-646 and Rattan et al, "Protein Synthesis:

Posttranslational Modifications and Aging", Ann NY Acad Sci (1992) 663:48- 62.

With respect to oligonucleotides, but not limited thereto, the term "specifically hybridizing" refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed "substantially complementary"). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.

A "coding sequence" or "coding region" refers to a nucleic acid molecule having sequence information necessary to produce a gene product, when the sequence is expressed.

Genes of the present invention may be isolated from appropriate biological sources using methods known in the art. In the exemplary embodiment of the invention, the genes may be isolated from genomic libraries of mouse. In alternative embodiments, cDNA clones may be isolated, such as been isolated from murine cDNA libraries. A preferred means for isolating the genes having the sequences set forth in Tables 1 and 2 is PCR amplification using genomic or cDNA templates and sequence specific primers. Genomic and cDNA libraries are commerically available, and can also be made by procedures well known in the art. In positions of degeneracy where more than one nucleic acid residue could be used to encode the appropriate amino acid residue, all the appropriate nucleic acid residues may be incorporated to create a mixed oligonucleotide population, or a neutral base such as inosine may be used. The strategy of oligonucleotide design is well known in the art.

Alternatively, PCR primers may be designed by the above method to match the coding sequences of a murine protein and these primers used to amplify the native nucleic acids from isolated cDNA or genomic DNA.

Hybridizations of the present invention may be performed, according to the method of Sambrook et al., using a hybridization solution comprising: 1.0% SDS, up to 50% formamide, 5x SSC (150mM NaCI, 15mM trisodium citrate), 0.05% sodium pyrophosphate (pH7.6), 5x Denhardt's solution, and 100 microgram/ml denatured, sheared salmon sperm DNA. Hybridization is carried out at 37-42°C for at least six hours. Following hybridization, filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) 30 minutes to 1 hour at 37°C in 2X SSC and 0.1% SDS; (4) 2 hours at 45-55 °C in 2X SSC and 0.1% SDS, changing the solution every 30 minutes.

One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified percent identity is set forth by (Sambrook et al, 1989, supra): T_m = 81.5 °C + 16.6Log [Na+] + 0.41(% G+C) - 0.63 (% formamide) - 600/#bp in duplex

As an illustration of the above formula, using [N+] = [0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the T_m is 57 °C. The T_m of a DNA duplex decreases by 1 - 1.5 °C with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42 °C.

The stringency of the hybridization and wash depend primarily on the salt concentration and temperature of the solutions. In general, to maximize the rate of annealing of the probe with its target, the hybridization is usually carried out at salt and temperature conditions that are 20 - 25 °C below the calculated T_m of the of the hybrid. Wash conditions should be as stringent as possible for the degree of identity of the probe for the target. In general, wash conditions are selected to be approximately 12 - 20 °C below the T_m of the hybrid. In regards to the nucleic acids of the current invention, a moderate stringency hybridization is defined as hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42 °C, and wash in 2X SSC and 0.5% SDS at 55 °C for 15 minutes. A high stringency hybridization is defined as hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42 °C, and wash in IX SSC and 0.5% SDS at 65 °C for 15 minutes. A very high stringency hybridization is defined as hybridization in 6X SSC, SX Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42 °C, and wash in 0.1 X SSC and 0.5% SDS at 65 °C for 15 minutes. As is well known in the art, hybridization stringencies may be adjusted to allow hybridization of nucleic acid probes with complementary sequences of varying degrees of homology.

Nucleic acids of the present invention may be maintained as DNA in any convenient cloning vector. In a preferred embodiment, clones are maintained in plasmid cloning/expression vector, such as pBluescript (Stratagene, La Jolla, CA), which is propagated in a suitable E. coli host cell.

The nucleic acid sequences of the invention are set forth in Tables 1 and 2. Tables 1 and 2 list publically available gene sequences and provides the appropriate Genbank Accession Numbers, the nucleotide and amino acid sequences of which are expressly incorporated by reference herein. Table 2 lists novel sequences which were isolated in the tissue screen described above. The genes of Table 2 are listed by their publically available TIGR Genbank Accession Numbers (see www.tigr.com). Based on the information provided in Tables 1 and 2, one of ordinary skill in the art can easily access these sequences for practicing the methods of the present invention. TABLE 1

Genes Highly Expressed in Prenatal Hippocampus (Cluster 1)

Mitosis/Cell CycleProgression

Accession No. Gene Name E16 P30 aa289122_s_at Cychn-dependent kinase regulatory subunit 2 17.21

X66032_s_at Cyclin B2 1 1.78 afu05885_s_at Cyclin G2 2.71

Msa 25099 0_s_at (sp Q04827) Gl/S-specific cyclin D2 2.98

L01640_s_at D-type Gl cyclin catalytic subunιt(PSK-J3/CDK4) 8.59

Msa.29759 0_f_at (sp P30285) Cell division protein kinase 4(PSK-J3) 1.51

M83749_s_at D-type cyclin (CYL2) 6 76

Msa 7498.0_s_at GADD45 (Growth arrest & DNA damage-induced protein) 2.07

AFO 1 1644_at Oral tumor suppressor homolog (Doc- 1 ) 1 1.13 01700-2_s_at p53 cellular tumor antigen 21.12

DNA and RNA Synthesis dl2513_f_at DNA topoisomerase II 21.89

X53068_s_at Proliferating cell nuclear antigen 2.1 1

D31966_s_at RNA polymerase I, 40 kD subunit 4 09

L25125 RNA hehcase & RNA-dependent ATPase from DEAD box 5.1 1

X63019_s_at U2-snRNPb (pRNPl l) 4 69

Msa.3346 0_s_at (sp P40070) U6-snRNA-assocιated protein 1 87

Msa.l8074.0_f_at Unwinding protein 1 8.99

Msa. l 8312.0_s_at pre-mRNA splicing factor SRP75 2.87 aa690583_at (gb: X70944) Myoblast cell surface antigen 24.1D5 3.91 aa269806_s_at (gb: Z35401) Histone H2A.X 4.37

M37736_f_at Replication-dependent histone H2A.1 13.03

X13171_s_at HI histone subtype HI (0) 1.88 aa285607_s_at (gb: M37583) Histone H2A.Z 8.85

Transcriptional Regulation xl7459_s_at J kappa RS-binding protein 5.76 aa030563_f_at (gb: M90356) Transcription factor BTF3 7.49

Msa.2969.0_s_at Neurogenιn-2 (ngn2) 8.44

AF004294_at Myelin transcription factor 1 4.58

X55316_s_at CAAT-box DNA binding protein subunit B (NF-YB) 3.07

U40825 s at WW-domain binding protein- 1 2.86

X04663_f_at Tubulin M-beta-5 482|

Chaperone Functions

Z31399_s_at CCT eta subunit (chaperonin containing TCP-1 ) 148

Z31555_s_at CCTepsilon subunit (chaperonin containing TCP-1 ) 158

Msa 5340_f_at CCT delta subunit (chaperonin containing TCP-1 ) 183

Z37164_s_at CCT theta subunit (cystohc chaperonin containing TCP-1 ; 213

Msa 7596 0_f_at (sp P4 8428) TCP 1 -chaperonin cofactor A 274

Protein Turnover

Msa 15493 0 _At (sp Valyl-tRNA synthetase 425

Msa 859200_s_at Threonyl-tRNA synthetase 273 Msa 20820_s_at Ubiquitin-conjugating enzyme (UbcM2) 301 Msa 66200_s_at Ubiquitin carboxyl-terminal hydrolase 439 Msa 216220_f_at Ubiquitin-activating enzyme El-X 224 u54803 Cysteine protease 2661

Cell Adhesion and Extracellular Matrix

J04694_s_at Collagen alpha- 1, type IV (col4a-l) 271

M18194_f_at Fibronectin (FN) 428

X56304_s_at Tenascin 4547

U20365_f_at Gamma-actin 168

X94310_s_at LI -like protein 231

Y00051_at Neural cell adhesion molecule (NCAM) 279^:

Msa 2554 0_at Neural cell adhesion molecule LI (NCAM-L1) 347

D50086_s_at Neurophi n 211 m31 131_s_at Neural cadheπn (N-cadheπn) 212

U54984 s at Membrane-type matπx metalloproteinase 1 301

Fatty Acid and Membrane Synthesis

U04827_s_at Brain fatty acid-binding protein (B-FABP) 865

X13135_s_at Fatty acid synthase 293 aa683731_s_at (gb M15856) Lipoprotein lipase precursor 226

D42048_s_at Squalene epoxidase 355

Msa 4964 0_f_at Famesyl pyrophosphate synthetase 252

X70100_f_at Keratinocyte pid-bindmg protein 416

U13262_s_at Myelin gene expression factor (MEF-2) 256 x83562 s at N-glycan alpha 2,8-sιalyltransferase 4894

Genes Up-regulated in Late Postnatal Stages (Clusters 11 & 15)

U66141_s_at Transcπption factor Sox-M 23.85

Glucose Metabolism

J03928 s_At Phosphofructokinase (PFK) 3.06

Msa.2087.0_f_at Pyruvate kinase 1.64

Msa.18475.0_f_at Glucose-6-phospate isomerase 6.35

AA717247_f_at (gb Y00516) Fructose bisphosphate aldolase A 6.58

Msa.l619.0_f_at Tnose phosphate isomerase 2.33

Msa.10491 0_s_at Gamma enolase (2-phospho-D-glycerate hydrolyase) 19.53

Msa.12494 )_f_at Alpha enolase (2-phospho-D-glycerate-hydro lyase 5.11 aa033394 at (SW- P00489) Glycogen phosphorylase 6.75 u48403 Glycerol kinase 1 55

Oxidative Metabolism aa415929_s_at NADH-ubiquinone oxidoreductase chain 49 KD 1.41 aa239003 s_at NADH-ubiquinone oxidoreductase AGGG subunit precursor 2

U37721_s_at Cytochrome c oxidase subunit VIII precursor (Cox81) 2.26

W53390_f_at Succinate Dehydrogenase 1.55

M29462 f at Malate dehydrogenase 2.63

X51905_f_at Lactate dehydrogenase - B 1.82

Msa.717.0_s_at Glycerophosphate dehydrogenase 124.51 af004670_s_at Antioxidant protein 2 (AOP2) 2.5

Membrane Regulation of Ionic Concentration x83933_s_at Ryanodine receptor type 2 32.61

Msa.l868.0_s_at Vacuolar Adenosine Triphosphatase, subunit B 2.16

Msal869.0_f_at Vacuolar Adenosine Triphosphatase, subunit E 2.29

U30840_s_at Voltage-dependent anion channel 1 1.35

Msa.l0561.0_s_at Sodium potassium transporting ATPase beta-1 chain 7.95

Msa.11196.0_at Sodium/potassium transporting ATPase alpha-2 chain 3.45

Msa.l871 .0_g_at Calcium-transporting ATPase sarcoplasmic reticulum type 2.46

Msa.23090.0_s_at Vacuolar ATP synthase subunit C 2.91

Msa.28655.0_s_at Vacuolar ATP synthase subunit AC45 5.96

Msa.29770.0_f_at Vacuolar ATP synthase 16 KD proteo lipid subunit 4.23

Msa.8313.0 s at Calcium-transporting ATPase endoplasmic reticulum type, 7.79 TABLE II

TC14224, TC14254, TC14312, TC14325, TC14329, TC14435.TC 14474,

TC14629, TC14635, TC14704, TC14731, TC14735, TC14762, TC14763,

TC14785, TC14788, TC14810, TC14823, TC14941, TC14972, TC14982,

TC15012, TC15118, TC15133, TC15141, TC15141, TC15204, TC15267,

TC15448, TC15584, TC15665, TC15831, TC15831, TC15974, TC16153,

TC16205, TC16355, TC16494, TC16651, TC16708, TC17122, TC17275,

TC17320, TC17874, TC17980, TC17980, TC18222, TC18241, TC18400,

TC18401, TC18687, TC18688, TC18708, TC18783, TC18804, TC18850,

TC18869, TC18884, TC19058, TC19062, TC19069, TC19085, TC19105,

TC19105, TC19136, TC19211, TC19521, TC19732, TC19823, TC19926,

TCI 9967, TC20081, TC20099, TC20539, TC20803, TC21082, TC21205,

TC21335, TC21412, TC21626, TC21685, TC21976, TC22202, TC22386,

TC22448, TC22529, TC22542, TC22668, TC22669, TC22696, TC22785,

TC23211, TC23244, TC23261, TC23542, TC23801, TC23956, TC24584,

TC26547, TC26624, TC26682, TC27097, TC27333, TC27344, TC27510,

TC27517, TC27528, TC27570, TC27571, TC27572, TC27573, TC27712,

TC27850, TC27894, TC27963, TC28142, TC28255, TC28416 TC28417,

TC28666, TC28824, TC28847, TC28859, TC28885, TC29042, TC29042,

TC29129, TC29216, TC29328, TC29385, TC29394, TC29445, TC29454,

TC29479, TC29708, TC29810, TC30188, TC30208, TC30378, TC30379,

TC30391, TC30530, TC30545, TC30555, TC30650, TC30755, TC30788, TC30805, TC30906, TC30918, TC30981, TC30987, TC31022, TC31051,

TC31091, TC31128, TC31250, TC31334, TC31339, TC31349, TC31386,

TC31671, TC31678, TC31686, TC31729, TC31755, TC31774, TC31783,

TC31827, TC31864, TC31882, TC31917, TC31921, TC32043, TC32074,

TC32106, TC32222, TC32250, TC32296, TC32304, TC32321, TC32325,

TC32339, TC32438, TC32456, TC32559, TC32602, TC32713, TC32808,

TC32829, TC32833, TC32980, TC33002, TC33009, TC33036, TC33177,

TC33178, TC33179, TC33208, TC33209, TC33231, TC33232, TC33244,

TC33290, TC33306, TC33377, TC33378, TC33384, TC33396, TC33407,

TC33529, TC33531, TC33738, TC33757, TC33765, TC33775, TC33788,

TC33816, TC33823, TC33849, TC33852, TC33859, TC33866, TC33882,

TC33882, TC33985, TC34265, TC34289, TC34379, TC34965, TC34983,

TC35017, TC35086, TC35131, TC35594, TC35648, TC35734, TC35822,

TC35823, TC35874, TC35937, TC35974, TC36080, TC36082, TC36142,

TC36344, TC36565, TC36683, TC36730, TC36740, TC36797, TC36816,

TC36917, TC36970, TC37016, TC37019, TC37101, TC37186, TC37226,

TC37230, TC37266, TC37268, TC37366, TC37388, TC37411, TC37468,

TC37472, TC37670, TC37689, TC37720, TC37721, TC37793, TC37793,

TC37904, TC38039, TC38045, TC38052, TC38091, TC38092, TC38136,

TC38142, TC38247, TC38281, TC38297, TC38377, TC38446, TC38523,

TC38552, TC38590, TC38627, TC38806, TC38862, TC38867, TC39079,

TC39101, TC39101, TC39196, TC39214, TC39296, TC39303, TC39305,

TC39334, TC39418, TC39420, TC39605, TC39644, TC39809, TC39826,

TC39827, TC39868, TC39877, TC39895, TC39990, TC40025, TC40265,

TC40450, TC40459, TC40494, TC40580, TC40603, TC40618, TC40687,

TC40689, TC40704, TC40734, TC40780, TC40817, TC40833, TC40840,

TC40879, TC40931, TC40975, TC41027, TC41069, TC41103, TC41175,

TC41197, TC41200, TC41472, TC41499, TC41551, TC41561, TC41569,

TC41588, TC41818, TC41859, TC41872, TC41992, and TC42157

EXAMPLES

Example 1

Matings and RNA Preparation: Timed matings were set up between

adult wild type mice from strain BCFl. Females were inspected for plugs on

the following day to ensure successful mating and the date of conception was

noted so that pups could be collected at the appropriate time.

Hippocampi were dissected from pups at the age of El 6, PI, P7, PI 6

and P30. The tissues were immediately frozen in liquid nitrogen, and then

stored at -80°C. Total RNA was isolated from tissue using the RNA

Extraction Kit (Pharmacia-Biotech), which involved extraction by

ultracentriftigation in a cesium triflouroacetate (CsTFA) gradient. Following

centrifugation, the supernatant was aspirated off, and then the RNA pellet at

the bottom of the tube was resuspended, followed by ethanol precipitation.

RNA concentration was determined spectrophotometrically by taking the optical density at 260 nm before processed further for Poly(A) RNA extraction

(Amersham Pharmacia-Biotech). Samples were stored at -80°C.

Example 2

Target Preparation: Double-stranded DNA was synthesized for each

target, using a Gibco BRL Superscript Choice System (Gibco BRL Life

Sciences, #18090-019). Typically, 1 μg of polyA RNA was used in a reverse

transcription reaction to make the first strand cDNA, utilizing a special oligo

primer containing poly-T and T7 RNA polymerase promoter sequences. The

double stranded cDNA was cleaned up using a phenol-chloroform extraction,

then ethanol-precipitated using glycogen as a carrier. The cDNA was

resuspended in 3 μl of RNase-free water. 1 μl of the double-stranded cDNA

was used as a template for the in vitro transcription (IVT) in the presence of

biotinylated UTP and CTP to generate high yield Blabeled antisense RNA.

The IVT reaction was performed using the Enzo BioArray High Yield RNA

Transcript Labeling Kit (Enzo Diagnostics, #900182). Clean-up of the IVT

reaction was performed using the Qiagen Rneasy Mini Kit spin columns

(Qiagen, #74104). The cleaned-up cRNA product of the IVT reaction was

quantified using spectrophotometric analysis at 260 nm.

Example 3

Array Hybridization and Scanning: The labeled cRNA was then

fragmented using a fragmentation buffer (5X buffer: 200 mM Tris-acetate, pH 8.1, 50 mM KOAc, 150 mM MgOAc), then was hybridized with chips in 200

microliter of hybridization solution containing 10 microgram labeled target in

IX MES buffer (0.1 M MES, 1.0 M NaCI, 0.01% Triton X-100, pH 6.7) and

O.lmg/ml herring sperm DNA. The arrays used in this study are Affymetrix

mouse expression arrays: 1 IK set and 19K set. Arrays were placed on a

rotisserie and rotated at 60 rpm for 16 hours at 45°C. Following hybridization,

the arrays were washed with 6X SSPE-T (0.9 M NaCI, 60 mM NaH2PO4, 6

mM EDTA, 0.005% Triton X-100, pH 7.6) at 22°C on a fluidics station

(Affymetrix) for 10X2 cycles, and then washed with 0.1 MES at 45°C for 30

min. The arrays were then stained with a streptavidin-phycoerythrin conjugate

(Molecular Probes), followed by 6X SSPE-T wash on the fluidics station for

10X2 cycles again. To enhance the signals, the arrays were further stained

with Anti-streptavidin antibody for 30 min followed by a 15 min staining with

a streptavidin-phycoerythrin conjugate again. After 6X SSPE-T wash on the

fluidics station for 10X2 cycles, the arrays were scanned at a resolution of 3

μm using a specifically designed confocal scanner (Affymetrix).

To further confirm the reliability of the array data, 16 genes were

randomly selected from the list, and subjected to reverse Northern blot

analysis. It was observed that these dynamic changes were consistent between

the two different methods. Image data was analyzed by GeneChip7k Analysis

Suite (Affymetrix). Gene clustering analysis was performed using

GeneCluster l.O (MIT).

Claims

CLAIMSWhat is claimed is:

1. A method of analyzing genome-wide molecular events occurring in brain tissue comprising the steps of hybridizing isolated brain mRNA to an oligonucleotide array; clustering groups of genes together using self-organizing map analysis; and analyzing alterations of expression levels of genes.

2. The method of claim 1 wherein the brain mRNA is hippocampal mRNA.

3. A brain development related nucleic acid molecule comprising a sequence selected from the group consisting of the accession numbers TC14224, TC14254, TC14312, TC14325, TC14329, TC14435/TC14474, TC14629, TC14635, TC14704, TC14731, TC14735, TC14762, TC14763, TC14785, TC14788, TC14810, TC14823, TC14941, TC14972, TC14982, TC15012, TC15118, TC15133, TC15141, TC15141, TC15204, TC15267, TC15448, TC15584, TC15665, TC15831, TC15831, TC15974, TC16153, TC16205, TC16355, TC16494, TC16651, TC16708, TC17122, TC17275, TC17320, TC17874, TC17980, TC17980, TC18222, TC18241, TC18400, TC18401, TC18687, TC18688, TC18708, TC18783, TC18804, TC18850, TC18869, TC18884, TC19058, TC19062, TC19069, TC19085, TC19105, TC19105, TC19136, TC19211, TC19521, TC19732, TC19823, TC19926, TC19967, TC20081, TC20099, TC20539, TC20803, TC21082, TC21205, TC21335, TC21412, TC21626, TC21685, TC21976, TC22202, TC22386, TC22448, TC22529, TC22542, TC22668, TC22669, TC22696, TC22785, TC23211, TC23244, TC23261, TC23542, TC23801, TC23956, TC24584, TC26547, TC26624, TC26682, TC27097, TC27333, TC27344, TC27510, TC27517, TC27528, TC27570, TC27571, TC27572, TC27573, TC27712, TC27850, TC27894, TC27963, TC28142, TC28255, TC28416 TC28417, TC28666, TC28824, TC28847, TC28859, TC28885, TC29042, TC29042, TC29129, TC29216, TC29328, TC29385, TC29394, TC29445, TC29454, TC29479, TC29708, TC29810, TC30188, TC30208, TC30378, TC30379, TC30391, TC30530, TC30545, TC30555, TC30650, TC30755, TC30788, TC30805, TC30906, TC30918, TC30981, TC30987, TC31022, TC31051, TC31091, TC31128, TC31250, TC31334, TC31339, TC31349, TC31386, TC31671, TC31678, TC31686, TC31729, TC31755, TC31774, TC31783, TC31827, TC31864, TC31882, TC31917, TC31921, TC32043, TC32074, TC32106, TC32222, TC32250, TC32296, TC32304, TC32321, TC32325, TC32339, TC32438, TC32456, TC32559, TC32602, TC32713, TC32808, TC32829, TC32833, TC32980, TC33002, TC33009, TC33036, TC33177, TC33178, TC33179, TC33208, TC33209, TC33231, TC33232, TC33244, TC33290, TC33306, TC33377, TC33378, TC33384, TC33396, TC33407, TC33529, TC33531, TC33738, TC33757, TC33765, TC33775, TC33788, TC33816, TC33823, TC33849, TC33852, TC33859, TC33866, TC33882, TC33882, TC33985, TC34265, TC34289, TC34379, TC34965, TC34983, TC35017, TC35086, TC35131, TC35594, TC35648, TC35734, TC35822, TC35823, TC35874, TC35937, TC35974, TC36080, TC36082, TC36142, TC36344, TC36565, TC36683, TC36730, TC36740, TC36797, TC36816, TC36917, TC36970, TC37016, TC37019, TC37101, TC37186, TC37226, TC37230, TC37266, TC37268, TC37366, TC37388, TC37411, TC37468, TC37472, TC37670, TC37689, TC37720, TC37721, TC37793, TC37793, TC37904, TC38039, TC38045, TC38052, TC38091, TC38092, TC38136, TC38142, TC38247, TC38281, TC38297, TC38377, TC38446, TC38523, TC38552, TC38590, TC38627, TC38806, TC38862, TC38867, TC39079, TC39101, TC39101, TC39196, TC39214, TC39296, TC39303, TC39305, TC39334, TC39418, TC39420, TC39605, TC39644, TC39809, TC39826, TC39827, TC39868, TC39877, TC39895, TC39990, TC40025, TC40265, TC40450, TC40459, TC40494, TC40580, TC40603, TC40618, TC40687, TC40689, TC40704, TC40734, TC40780, TC40817, TC40833, TC40840, TC40879, TC40931, TC40975, TC41027, TC41069, TC41103, TC41175, TC41197, TC41200, TC41472, TC41499, TC41551, TC41561, TC41569, TC41588, TC41818, TC41859, TC41872, TC41992, and TC42157.

90

4. A method for isolating protein sequences, comprising: a) assembling a hybridization reaction mixture containing one or more or the isolated nucleic acid molecules in single stranded form, and a test sample that comprises the corresponding protein coding sequence, in a

95 single-stranded form, under conditions enabling hybridization of the isolated nucleic acid molecule and the protein sequence, thereby forming a double- stranded nucleic acid molecule; b) separating the double stranded molecule comprising the isolated nucleic acid and the protein coding sequence; and

100 c) cloning the protein coding sequence.

5. An isolated protein produced by expression of the protein coding sequences isolated by the method of claim 4.

105 6. Antibodies immunologically specific for the protein molecules of claim 5.

7. A recombinant DNA molecule comprising a protein coding sequence produced by the method of claim 4; operably linked to a vector for

110 transforming cells.

8. A method of screening for candidate drugs which induce or inhibit expression of genes in the hippocampus comprising the steps of: contacting a hippocampal cell with a candidate drug for 115 sufficient time for detectable expression of a gene; and assaying for the amount of expression in the cell of two or more genes selected from the group consisting of Cyclin-dependent kinase regulatory subunit 2, Cyclin B2, Cyclin G2, Gl/S-specific cyclin D2, D-type Gl cyclin catalytic subunit(PSK-J3/CDK4), Cell division protein kinase

120 4(PSK- J3), D-type cyclin (C YL2), GADD45 (Growth arrest & DNA damage- induced protein), Oral tumor suppressor homolog (Doc-1), p53 cellular tumor antigen, DNA topoisomerase II, Proliferating cell nuclear antigen, RNA polymerase I, 40 kD subunit, RNA helicase & RNA-dependent ATPase from DEAD box family, U2-snRNPb (pRNPl 1), U6-snRNA-associated protein, 125 Unwinding protein 1, pre-mRNA splicing factor SRP75, Myoblast cell surface antigen 24.1D5, Histone H2A.X, Replication-dependent histone H2A.1, HI histone subtype HI (0), Histone H2A.Z, J kappa RS-binding protein , Transcription factor BTF3, Neurogenin-2 (ngn2) , Myelin transcription factor 1, CAAT-box DNA binding protein subunit B (NF-YB), WW-domain binding

130 protein- 1 , Elongation factor- 1 -alpha, Elongation factor- 1 -gamma, Elongation factor-2(EF-2), Iniatiation Factor 2 associated 67KD glycoprotein, Translational initiation factor 2 beta subunit (EIF-2), Ubiquitin-conjugating enzyme E2, 60S ribosomal protein A52, Ribosomal proteinL5, Ribosomal protein L8 (RPL8), 60S ribosomal protein L9, Ribosomal protein LI 2 135 (RPL12), 60S ribosomal protein L13A, Ribosomal protein L18 (RPL18),

Ribosomal protein LI 9, Ribosomal protein L30 , 60S ribosomal protein L23, 60S ribosomal protein L32, 60S ribosomal protein L34, 60S ribosomal protein L37, Ribosomal protein S8, 40S ribosomal protein S24, Ribosomal protein Ke-3, Actin, cytoplasmic 1 (beta actin), Actin- 1, Actin-3, Cytoskeletal gamma

140 actin , Tubulin M-alpha-3, Tubulin beta-1 chain, Tubulin beta-2 chain,

Tubulin alpha-1, Tubulin alpha-4,Tubulin alpha-5, Tubulin alpha-6, Tubulin alpha-8, Tubulin beta-3, Tubulin beta-4(classHI), Tubulin M-alpha-2, Tubulin M-alpha-3, Tubulin M-beta-5, CCT eta subunit (chaperonin containing TCP- 1), CCTepsilon subunit (chaperonin containing TCP-1), CCT delta subunit 145 (chaperonin containing TCP- 1 ), CCT theta subunit (cystolic chaperonin containing TCP-1), TCP 1 -chaperonin cofactor A, Valyl-tRNA synthetase, Threonyl-tRNA synthetase, Ubiquitin-conjugating enzyme (UbcM2), Ubiquitin carboxyl-terminal hydrolase, Ubiquitin-activating enzyme El-X, Cysteine protease, Collagen alpha- 1, type IV (col4a-l), Fibronectin (FN),

150 Tenascin, Gamma-actin, LI -like protein , Neural cell adhesion molecule

(NCAM), Neural cell adhesion molecule LI (NCAM-L1), Neurophilin, Neural cadherin (N-cadherin), Membrane-type matrix metalloproteinase 1, Brain fatty acid-binding protein (B-FABP), Fatty acid synthase, Lipoprotein lipase precursor, Squalene epoxidase, Famesyl pyrophosphate synthetase,

155 Keratinocyte lipid-binding protein, Myelin gene expression factor (MEF-2), N- glycan alpha 2,8-sialyltransferase, Clathrin, light chain B, Synaptogamin 1/65, UNC-18 homologue, Vesicle associated membrane protein (VAMP2), Synaptophysin (major synaptic vesicle protein P38), Neuronal pentraxin 1, N- methyl-D-aspartate receptor - glutamate binding chain , Neurogranin (protein

160 kinase C substrate 7.5 kD), alpha-SNAP protein, Calcium-activated potassium channel, Potassium channel, beta subunit, Brain neurotensin receptor, Acetylcholine receptor-interacting protein (AIP), Fractalkine, Cholecystokinin, Brain derived neurotrophic factor (BDNF), Glutamate receptor 1 (GluRl), Glutamate receptor 2 (GluR2), SH3 -containing protein (SH3P4), SH3P9, C-H-

165 Ras, Ras-related protein RAB-3 A , Mitogen activated protein kinase (erk-1), Receptor-type tyrosine kinase, Focal adhesion kinase, Calcineurin, Phospholipase C beta 1, Diglyceride kinase, RhoB, Protein kinase C, Cell division cycle homolog (CDC25), Elongation factor- l(alpha-CMSl), Growth factor-induced protein (zif/268), DNA-binding protein (Smbp-2), Proto-

170 oncogene DBL, Transcriptional activator FE6J, Cell division control protein 4, Transcription factor Sox-M, Phosphofructokinase (PFK), Pyruvate kinase, Glucose-6-phospate isomerase, Fructose bisphosphate aldolase A, Triose phosphate isomerase, Gamma enolase (2-phospho-D-glycerate hydrolyase), Alpha enolase (2-phospho-D-glycerate-hydrolyase, Glycogen phosphorylase,

175 Glycerol kinase, NADH-ubiquinone oxidoreductase chain 49 KD subunit, NADH-ubiquinone oxidoreductase AGGG subunit precursor, Cytochrome c oxidase subunit VHI precursor (Cox81), Succinate Dehydrogenase, Malate dehydrogenase, Lactate dehydrogenase - B, Glycerophosphate dehydrogenase, Antioxidant protein 2 (AOP2), Ryanodine receptor type 2, Vacuolar

180 Adenosine Triphosphatase, subunit B, Vacuolar Adenosine Triphosphatase, subunit E, Voltage-dependent anion channel 1, Sodium/potassium transporting ATPase beta-1 chain, Sodium/potassium transporting ATPase alpha-2 chain, Calcium-transporting ATPase sarcoplasmic reticulum type, Vacuolar ATP synthase subunit C, Vacuolar ATP synthase subunit AC45, Vacuolar ATP

185 synthase 16 KD proteolipid subunit, and Calcium-transporting ATPase endoplasmic reticulum type class 2, wherein the expression of said genes in the cell is assayed before and after the cell has been contacted with the test substance, and wherein the candidate drug is identified if it increases or decreases expression of one or more of said genes.

190

9. A method of treating a disease comprising administering to a diseased patient a therapeutically effective amount of a gene comprising a sequence selected from the group consisting of Cyclin-dependent kinase regulatory subunit 2, Cyclin B2, Cyclin G2, Gl/S-specific cyclin D2, D-type

195 Gl cyclin catalytic subunit(PSK-J3/CDK4), Cell division protein kinase

4(PSK-J3), D-type cyclin (CYL2), GADD45 (Growth arrest & DNA damage- induced protein), Oral tumor suppressor homolog (Doc-1), p53 cellular tumor antigen, DNA topoisomerase π, Proliferating cell nuclear antigen, RNA polymerase I, 40 kD subunit, RNA helicase & RNA-dependent ATPase from

200 DEAD box family, U2-snRNPb (pRNP 11 ), U6-snRNA-associated protein,

Unwinding protein 1, pre-mRNA splicing factor SRP75, Myoblast cell surface antigen 24.1D5, Histone H2A.X, Replication-dependent histone H2A.1, HI histone subtype HI (0), Histone H2A.Z, J kappa RS-binding protein , Transcription factor BTF3, Neurogenin-2 (ngn2) , Myelin transcription factor

205 1 , C AAT-box DNA binding protein subunit B (NF-YB), WW-domain binding protein- 1, Elongation factor- 1 -alpha, Elongation factor- 1 -gamma, Elongation factor-2(EF-2), Iniatiation Factor 2 associated 67KD glycoprotein, Translational initiation factor 2 beta subunit (EIF-2), Ubiquitin-conjugating enzyme E2, 60S ribosomal protein A52, Ribosomal proteinL5, Ribosomal 210 protein L8 (RPL8), 60S ribosomal protein L9, Ribosomal protein L12

(RPL12), 60S ribosomal protein LI 3 A, Ribosomal protein LI 8 (RPL18), Ribosomal protein LI 9, Ribosomal protein L30 , 60S ribosomal protein L23, 60S ribosomal protein L32, 60S ribosomal protein L34, 60S ribosomal protein L37, Ribosomal protein S8, 40S ribosomal protein S24, Ribosomal protein

215 Ke-3, Actin, cytoplasmic 1 (beta actin), Actin-1, Actin-3, Cytoskeletal gamma actin , Tubulin M-alpha-3, Tubulin beta-1 chain, Tubulin beta-2 chain, Tubulin alpha- 1, Tubulin alpha-4,Tubulin alpha-5, Tubulin alpha-6, Tubulin alpha-8, Tubulin beta-3, Tubulin beta-4(classIII), Tubulin M-alpha-2, Tubulin M-alpha-3, Tubulin M-beta-5, CCT eta subunit (chaperonin containing TCP-

220 1), CCTepsilon subunit (chaperonin containing TCP-1), CCT delta subunit (chaperonin containing TCP-1), CCT theta subunit (cystolic chaperonin containing TCP-1), TCP 1 -chaperonin cofactor A, Valyl-tRNA synthetase, Threonyl-tRNA synthetase, Ubiquitin-conjugating enzyme (UbcM2), Ubiquitin carboxyl-terminal hydrolase, Ubiquitin-activating enzyme El-X,

225 Cysteine protease, Collagen alpha- 1 , type IV (col4a- 1 ), Fibronectin (FN),

Tenascin, Gamma- actin, LI -like protein , Neural cell adhesion molecule (NCAM), Neural cell adhesion molecule LI (NCAM-L1), Neurophilin, Neural cadherin (N-cadherin), Membrane-type matrix metalloproteinase 1, Brain fatty acid-binding protein (B-FABP), Fatty acid synthase, Lipoprotein lipase

230 precursor, Squalene epoxidase, Famesyl pyrophosphate synthetase,

Keratinocyte lipid-binding protein, Myelin gene expression factor (MEF-2), N- glycan alpha 2,8-sialyltransferase, Clathrin, light chain B, Synaptogamin 1 65, UNC-18 homologue, Vesicle associated membrane protein (VAMP2), Synaptophysin (major synaptic vesicle protein P38), Neuronal pentraxin 1, N-

235 methyl-D-aspartate receptor - glutamate binding chain , Neurogranin (protein kinase C substrate 7.5 kD), alpha-SNAP protein, Calcium-activated potassium channel, Potassium channel, beta subunit, Brain neurotensin receptor, Acetylcholine receptor-interacting protein (AJP), Fractalkine, Cholecystokinin, Brain derived neuro trophic factor (BDNF), Glutamate receptor 1 (GluRl),

240 Glutamate receptor 2 (GluR2), SH3 -containing protein (SH3P4), SH3P9, C-H- Ras, Ras-related protein RAB-3 A , Mitogen activated protein kinase (erk-1), Receptor-type tyrosine kinase, Focal adhesion kinase, Calcineurin, Phospholipase C beta 1 , Diglyceride kinase, RhoB, Protein kinase C, Cell division cycle homolog (CDC25), Elongation factor- l(alpha-CMSl), Growth

245 factor-induced protein (zif/268), DNA-binding protein (Smbp-2), Proto- oncogene DBL, Transcriptional activator FE6J, Cell division control protein 4, Transcription factor Sox-M, Phosphofructokinase (PFK), Pyruvate kinase, Glucose-6-phospate isomerase, Fructose bisphosphate aldolase A, Triose phosphate isomerase, Gamma enolase (2-phospho-D-glycerate hydrolyase),

250 Alpha enolase (2-phospho-D-glycerate-hydrolyase, Glycogen phosphorylase, Glycerol kinase, NADH-ubiquinone oxidoreductase chain 49 KD subunit, NADH-ubiquinone oxidoreductase AGGG subunit precursor, Cytochrome c oxidase subunit VEQ precursor (Cox81), Succinate Dehydrogenase, Malate dehydrogenase, Lactate dehydrogenase - B, Glycerophosphate dehydrogenase,

255 Antioxidant protein 2 (AOP2), Ryanodine receptor type 2, Vacuolar

Adenosine Triphosphatase, subunit B, Vacuolar Adenosine Triphosphatase, subunit E, Voltage-dependent anion channel 1, Sodium/potassium transporting ATPase beta-1 chain, Sodium/potassium transporting ATPase alpha-2 chain, Calcium-transporting ATPase sarcoplasmic reticulum type, Vacuolar ATP

260 synthase subunit C, Vacuolar ATP synthase subunit AC45, Vacuolar ATP synthase 16 KD proteolipid subunit, and Calcium-transporting ATPase endoplasmic reticulum type class 2; or administering to a diseased patient a therapeutically effective amount of a polypeptide that competes with the polypeptide encoded

265 by any one of said genes for its ligand, substrate, or receptor.

10. A method of treating a disease comprising administering to a diseased patient a therapeutically effective amount of a gene comprising a sequence selected from the group consisting of accession numbers an isolated 270 nucleic acid molecule comprising a nucleotide sequence as set forth in an any one of the accession numbers TCI 4224, TCI 4254, TCI 4312, TCI 4325, TC14329, TC14435 C14474, TC14629, TC14635, TC14704, TC14731, TC14735, TC14762, TC14763, TC14785, TC14788, TC14810, TC14823, TC14941, TC14972, TC14982, TC15012, TC15118, TC15133, TC15141,

275 TC15141, TC15204, TC15267, TC15448, TC15584, TC15665, TC15831, TC15831, TC15974, TC16153, TC16205, TC16355, TC16494, TC16651, TC16708, TC17122, TC17275, TC17320, TC17874, TC17980, TC17980, TC18222, TC18241, TC18400, TC18401, TC18687, TC18688, TC18708, TC18783, TC18804, TC18850, TC18869, TC18884, TC19058, TC19062,

280 TC19069, TC19085, TC19105, TC19105, TC19136, TC19211, TC19521, TC19732, TC19823, TC19926, TC19967, TC20081, TC20099, TC20539, TC20803, TC21082, TC21205, TC21335, TC21412, TC21626, TC21685, TC21976, TC22202, TC22386, TC22448, TC22529, TC22542, TC22668, TC22669, TC22696, TC22785, TC23211, TC23244, TC23261, TC23542,

285 TC23801, TC23956, TC24584, TC26547, TC26624, TC26682, TC27097, TC27333, TC27344, TC27510, TC27517, TC27528, TC27570, TC27571, TC27572, TC27573, TC27712, TC27850, TC27894, TC27963, TC28142, TC28255, TC28416 TC28417, TC28666, TC28824, TC28847, TC28859, TC28885, TC29042, TC29042, TC29129, TC29216, TC29328, TC29385,

290 TC29394, TC29445, TC29454, TC29479, TC29708, TC29810, TC30188, TC30208, TC30378, TC30379, TC30391, TC30530, TC30545, TC30555, TC30650, TC30755, TC30788, TC30805, TC30906, TC30918, TC30981, TC30987, TC31022, TC31051, TC31091, TC31128, TC31250, TC31334, TC31339, TC31349, TC31386, TC31671, TC31678, TC31686, TC31729,

295 TC31755, TC31774, TC31783, TC31827, TC31864, TC31882, TC31917, TC31921, TC32043, TC32074, TC32106, TC32222, TC32250, TC32296, TC32304, TC32321, TC32325, TC32339, TC32438, TC32456, TC32559, TC32602, TC32713, TC32808, TC32829, TC32833, TC32980, TC33002, TC33009, TC33036, TC33177, TC33178, TC33179, TC33208, TC33209,

300 TC33231, TC33232, TC33244, TC33290, TC33306, TC33377, TC33378, TC33384, TC33396, TC33407, TC33529, TC33531, TC33738, TC33757, TC33765, TC33775, TC33788, TC33816, TC33823, TC33849, TC33852, TC33859, TC33866, TC33882, TC33882, TC33985, TC34265, TC34289, TC34379, TC34965, TC34983, TC35017, TC35086, TC35131, TC35594,

305 TC35648, TC35734, TC35822, TC35823, TC35874, TC35937, TC35974, TC36080, TC36082, TC36142, TC36344, TC36565, TC36683, TC36730, TC36740, TC36797, TC36816, TC36917, TC36970, TC37016, TC37019, TC37101, TC37186, TC37226, TC37230, TC37266, TC37268, TC37366, TC37388, TC37411, TC37468, TC37472, TC37670, TC37689, TC37720,

310 TC37721, TC37793, TC37793, TC37904, TC38039, TC38045, TC38052, TC38091, TC38092, TC38136, TC38142, TC38247, TC38281, TC38297, TC38377, TC38446, TC38523, TC38552, TC38590, TC38627, TC38806, TC38862, TC38867, TC39079, TC39101, TC39101, TC39196, TC39214, TC39296, TC39303, TC39305, TC39334, TC39418, TC39420, TC39605,

315 TC39644, TC39809, TC39826, TC39827, TC39868, TC39877, TC39895, TC39990, TC40025, TC40265, TC40450, TC40459, TC40494, TC40580, TC40603, TC40618, TC40687, TC40689, TC40704, TC40734, TC40780, TC40817, TC40833, TC40840, TC40879, TC40931, TC40975, TC41027, TC41069, TC41103, TC41175, TC41197, TC41200, TC41472, TC41499,

320 TC41551, TC41561, TC41569, TC41588, TC41818, TC41859, TC41872, TC41992, or TC42157; or administering to a diseased patient a therapeutically effective amount of a polypeptide that competes with the polypeptide encoded by any one of said sequences for its ligand, substrate, or receptor.

325

11. A method of screening for candidate drugs which induce or inhibit expression of genes in the hippocampus comprising the steps of: contacting a hippocampal cell with a candidate drug for sufficient time for detectable expression of a gene; and

330 assaying for the amount of expression in the cell of two or more genes selected from the group consisting of the accession numbers TCI 4224, TC14254, TC14312, TC14325, TC14329, TC14435 C14474, TC14629, TC14635, TC14704, TC14731, TC14735, TC14762, TC14763, TC14785, TC14788, TC14810, TC14823, TC14941, TC14972, TC14982, TC15012,

335 TC15118, TC15133, TC15141, TC15141, TC15204, TC15267, TC15448, TC15584, TC15665, TC15831, TC15831, TC15974, TC16153, TC16205, TC16355, TC16494, TC16651, TC16708, TC17122, TC17275, TC17320, TC17874, TC17980, TC17980, TC18222, TC18241, TC18400, TC18401, TC18687, TC18688, TC18708, TC18783, TC18804, TC18850, TC18869,

340 TC18884, TC19058, TC19062, TC19069, TC19085, TC19105, TC19105, TC19136, TC19211, TC19521, TC19732, TC19823, TC19926, TC19967, TC20081 , TC20099, TC20539, TC20803, TC21082, TC21205, TC21335, TC21412, TC21626, TC21685, TC21976, TC22202, TC22386, TC22448, TC22529, TC22542, TC22668, TC22669, TC22696, TC22785, TC23211,

345 TC23244, TC23261, TC23542, TC23801, TC23956, TC24584, TC26547, TC26624, TC26682, TC27097, TC27333, TC27344, TC27510, TC27517, TC27528, TC27570, TC27571, TC27572, TC27573, TC27712, TC27850, TC27894, TC27963, TC28142, TC28255, TC28416 TC28417, TC28666, TC28824, TC28847, TC28859, TC28885, TC29042, TC29042, TC29129,

350 TC29216, TC29328, TC29385, TC29394, TC29445, TC29454, TC29479, TC29708, TC29810, TC30188, TC30208, TC30378, TC30379, TC30391, TC30530, TC30545, TC30555, TC30650, TC30755, TC30788, TC30805, TC30906, TC30918, TC30981, TC30987, TC31022, TC31051, TC31091, TC31128, TC31250, TC31334, TC31339, TC31349, TC31386, TC31671,

355 TC31678, TC31686, TC31729, TC31755, TC31774, TC31783, TC31827, TC31864, TC31882, TC31917, TC31921, TC32043, TC32074, TC32106, TC32222, TC32250, TC32296, TC32304, TC32321, TC32325, TC32339, TC32438, TC32456, TC32559, TC32602, TC32713, TC32808, TC32829, TC32833, TC32980, TC33002, TC33009, TC33036, TC33177, TC33178,

360 TC33179, TC33208, TC33209, TC33231 , TC33232, TC33244, TC33290, TC33306, TC33377, TC33378, TC33384, TC33396, TC33407, TC33529, TC33531, TC33738, TC33757, TC33765, TC33775, TC33788, TC33816, TC33823, TC33849, TC33852, TC33859, TC33866, TC33882, TC33882, TC33985, TC34265, TC34289, TC34379, TC34965, TC34983, TC35017, 365 TC35086, TC35131, TC35594, TC35648, TC35734, TC35822, TC35823, TC35874, TC35937, TC35974, TC36080, TC36082, TC36142, TC36344, TC36565, TC36683, TC36730, TC36740, TC36797, TC36816, TC36917, TC36970, TC37016, TC37019, TC37101, TC37186, TC37226, TC37230, TC37266, TC37268, TC37366, TC37388, TC37411, TC37468, TC37472,

370 TC37670, TC37689, TC37720, TC37721, TC37793, TC37793, TC37904, TC38039, TC38045, TC38052, TC38091, TC38092, TC38136, TC38142, TC38247, TC38281, TC38297, TC38377, TC38446, TC38523, TC38552, TC38590, TC38627, TC38806, TC38862, TC38867, TC39079, TC39101, TC39101, TC39196, TC39214, TC39296, TC39303, TC39305, TC39334,

375 TC39418, TC39420, TC39605, TC39644, TC39809, TC39826, TC39827, TC39868, TC39877, TC39895, TC39990, TC40025, TC40265, TC40450, TC40459, TC40494, TC40580, TC40603, TC40618, TC40687, TC40689, TC40704, TC40734, TC40780, TC40817, TC40833, TC40840, TC40879, TC40931, TC40975, TC41027, TC41069, TC41103, TC41175, TC41197,

380 TC41200, TC41472, TC41499, TC41551, TC41561, TC41569, TC41588, TC41818, TC41859, TC41872, TC41992, and TC42157, wherein the expression of said genes in the cell is assayed before and after the cell has been contacted with the test substance, and wherein the candidate drug is identified if it increases or decreases expression of one or more of said genes.