EP1226280A2 - Sequences genetiques associees a la proliferation et aux pathologies des cellules neuronales - Google Patents

Sequences genetiques associees a la proliferation et aux pathologies des cellules neuronales

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Publication number
EP1226280A2
EP1226280A2 EP00988496A EP00988496A EP1226280A2 EP 1226280 A2 EP1226280 A2 EP 1226280A2 EP 00988496 A EP00988496 A EP 00988496A EP 00988496 A EP00988496 A EP 00988496A EP 1226280 A2 EP1226280 A2 EP 1226280A2
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European Patent Office
Prior art keywords
protein
subunit
alpha
genes
tubulin
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EP00988496A
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German (de)
English (en)
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EP1226280A4 (fr
Inventor
Joe Z. Tsien
Monica Princeton Univ. Dept. Molecular Bio. MODY
Yanxiang Cao
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Affymetrix Inc
Princeton University
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Affymetrix Inc
Princeton University
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Publication of EP1226280A2 publication Critical patent/EP1226280A2/fr
Publication of EP1226280A4 publication Critical patent/EP1226280A4/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the mRNA is hippocampal mRNA.
  • the mRNA is comprised of polynucleotide sequences of Tables 1 or 2.
  • 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.
  • 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.
  • 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.
  • 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.
  • SOM self-organizing map
  • 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.
  • 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.
  • 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.
  • GeneChip7k permits scanning significant portions of the genome and profiling of comprehensive molecular and genetic programs underlying the mammalian brain development.
  • 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.
  • SOM self-organizing map analysis
  • 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 ⁇ , 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.
  • 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.
  • Png-1 Postmitotic Neural Gene-1
  • Type IV includes the clusters C2, C3 and C6, and exhibits down- regulation at PI or P7.
  • 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.
  • 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.
  • 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.
  • penetrants such as bile salts or fusidic acids or other detergents.
  • 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 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.
  • 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.
  • solid phase arrays are used for the rapid and specific detection of nucleic acid molecules and their expression patterns.
  • 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.
  • a target nucleic acid e.g., a genomic nucleic acid, an amplicon, or, most commonly, an amplified mixture
  • Either the probe, or the target, or both can be labeled, typically with a fluorophore or other tag, such as streptavidin.
  • hybridization is detected by detecting bound fluorescence.
  • hybridization is typically detected by quenching of the label.
  • detection of hybridization is typically performed by monitoring a color shift resulting from proximity of the two bound labels.
  • Probes of the present invention are synthesized on a solid support.
  • Exemplary solid supports include glass, plastics, polymers, metals, metalloids, ceramics, organics, etc.
  • 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 cm 2 to several cm 2 , thereby incorporating sets of from a few to millions of probes.
  • 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.
  • 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).
  • 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.
  • 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.
  • RNA polymerase II 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.
  • RNA secondary structure 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.
  • RNA polymerase II a transcription factor required for transcription initiation of RNA polymerase II
  • NF1-B a transcription factor required for transcription initiation of RNA polymerase II
  • NF1-B a transcription factor required for transcription initiation of RNA polymerase II
  • NF1-B a transcription factor required for transcription initiation of RNA polymerase II
  • NF1-B a transcription factor required for transcription initiation of RNA polymerase II
  • NF1-B which mediates the transcription of several differentiation markers
  • 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.
  • 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).
  • 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.
  • 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.
  • cell adhesion and extracellular matrix proteins are also up-regulated during the neonatal and early postnatal period. 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.
  • 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.
  • 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.
  • clathrin which is a component of the coat that surrounds vesicles and synaptogamin, a vesicle-associated protein involved in calcium-mediated release of neurotransmitter.
  • VAMP2 and synaptophysin 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).
  • chemokine fractalkine (Meucci O. et al. (1998) PNAS USA 95, 14500- 14505), cholecystokinin (Kelly JS et al (1981) Adv. Biochem.
  • BDNF brain-derived neurotrophic factor
  • BDNF brain-derived neurotrophic factor
  • 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).
  • GluRl glutamate receptor 1
  • GluR2 glutamate receptor 2
  • AP acetylcholine
  • the neurotensin receptor was identified (Vincent JP, et al. (1999) Trends Pharmacol. Sci. 20, 302-309).
  • transcriptional and translational factors were 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.
  • 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 Fearaonio R. et al. (1994) Nucleic Acids Res. 22, 4876-4883
  • Sox-M were identified.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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 - STRUC
  • 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”).
  • 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.
  • the genes may be isolated from genomic libraries of mouse.
  • 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.
  • nucleic acid residues may be incorporated to create a mixed oligonucleotide population, or a neutral base such as inosine may be used.
  • a neutral base such as inosine
  • 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.
  • 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.
  • 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.
  • a moderate stringency hybridization is defined as hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 ⁇ g/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 ⁇ g/ml denatured salmon sperm DNA at 42 °C, and wash in IX SSC and 0.5% SDS at 65 °C for 15 minutes.
  • 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.
  • 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.
  • 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
  • NCAM Neural cell adhesion molecule
  • Oxidative Metabolism aa415929_s_at NADH-ubiquinone oxidoreductase chain 49 KD 1.41 aa239003 s_at NADH-ubiquinone oxidoreductase AGGG subunit precursor 2
  • TC33178 TC33179, TC33208, TC33209, TC33231, TC33232, TC33244,
  • Hippocampi were dissected from pups at the age of El 6, PI, P7, PI 6
  • CsTFA cesium triflouroacetate
  • RNA concentration was determined spectrophotometrically by taking the optical density at 260 nm before processed further for Poly(A) RNA extraction
  • Double-stranded DNA was synthesized for each
  • double stranded cDNA was cleaned up using a phenol-chloroform extraction
  • the IVT reaction was performed using the Enzo BioArray High Yield RNA
  • fragmented using a fragmentation buffer (X buffer: 200 mM Tris-acetate, pH 8.1, 50 mM KOAc, 150 mM MgOAc), then was hybridized with chips in 200
  • IX MES buffer 0.1 M MES, 1.0 M NaCI, 0.01% Triton X-100, pH 6.7
  • mouse expression arrays 1 IK set and 19K set. Arrays were placed on a
  • Image data was analyzed by GeneChip7k Analysis

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Abstract

Par l'application à grande échelle de techniques reposant sur l'utilisation de micropuces à ADN, les programmes du développement moléculaire de l'hippocampe, une structure corticale essentielle pour l'apprentissage et la mémoire, ont été analysés. Plus de 30 000 gènes et séquences EST ont été criblés, notamment une proportion estimée à 30-40 % des gènes totaux du génome de la souris. La présente invention porte sur l'identification de 4 390 gènes qui ont montré des modifications dynamiques durant le développement de l'hippocampe. A l'aide d'une analyse par batteries de gènes, ces gènes ont en outre été regroupés en 16 batteries distinctes présentant des structures particulières pouvant être mises en corrélation avec les principaux repères du développement. On a procédé ensuite à l'analyse de la covariance, qui a permis d'identifier un groupe de gènes dont l'expression pourrait être à l'origine des modifications phénotypiques dans l'hippocampe.
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