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EP1282701A2 - Nouvelle proteine a expression neuronale et son utilisation - Google Patents

Nouvelle proteine a expression neuronale et son utilisation

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Publication number
EP1282701A2
EP1282701A2 EP01927914A EP01927914A EP1282701A2 EP 1282701 A2 EP1282701 A2 EP 1282701A2 EP 01927914 A EP01927914 A EP 01927914A EP 01927914 A EP01927914 A EP 01927914A EP 1282701 A2 EP1282701 A2 EP 1282701A2
Authority
EP
European Patent Office
Prior art keywords
seq
protein
nucleic acid
acid sequence
lloo
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01927914A
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German (de)
English (en)
Inventor
Bernhard Götz
Birgitta Kammandel
Rohini Kuner
Sigrid Scheek
Holger Hiemisch
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sygnis Pharma AG
Original Assignee
BASF Lynx Bioscience AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by BASF Lynx Bioscience AG filed Critical BASF Lynx Bioscience AG
Publication of EP1282701A2 publication Critical patent/EP1282701A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • 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
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to new, specifically expressed proteins, and nucleic acid sequences or recombinant nucleic acid constructs which code for the proteins.
  • the invention also relates to host organisms or transgenic animals containing the nucleic acid sequences or recombinant nucleic acid constructs and mono- or polyclonal antibodies which are directed against the isolated proteins.
  • the invention further relates to a method for finding substances with a specific binding affinity for the proteins according to the invention, a method for the qualitative or quantitative detection of the nucleic acid sequences according to the invention or the proteins according to the invention.
  • the invention further relates to the use of the nucleic acid sequences and proteins.
  • IEG Homer 1A For the IEG Homer 1A, for example, it could be shown that this is a truncated variant of a member of a larger gene family and that the induction of this variant leads to a (dominant-negative) interruption of signal transmission, which is different from the other members of the gene family extracellular receptors and internal calcium stores is mediated [Tu et al. , (1998) Neuron 21: 717-726; 73; Xiao et al. , (1998) Neuron 21: 707-716; Yuguchi et al. , (1997), J. Cereb. Blood flow metab. 17: 745 - 752.
  • an external stimulus leads to direct changes in important second messenger systems.
  • IEGs neuron-expressed IEGs in the hippocampus
  • This gene is also induced in its mRNA expression after a seizure has been triggered in pyramidal cells of the hippocampal subregions CA1 and CA3. It has been shown that Are mRNA expression in the CAl area is induced simply by moving the rat into a new environment. Since the pattern of the induced neurons was specific for the respective environment in which the rat was located, it was possible to demonstrate that the induction of the Are mRNA expression correlates with processes of neuronal information storage in the hippocampus [Guzowski et al., (1999 ), Nat. Neurosci. 2: 1120-1124]. These so-called IEGs appear to represent important intracellular regulatory points. They play an important role in the development of organisms and in pathological processes within the organism or within the individual cell.
  • nucleic acid sequences which, as a result of the degenerate genetic code, are derived from the nucleic acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 8,
  • nucleic acids can advantageously be found in eukaryotic and prokaryotic organisms such as mammals such as Homo sapiens, Rattus, norvegicus or Mus musculus. These nucleic acids code for the amino acid sequences SEQ ID NO: 2 (Homo sapiens), SEQ ID NO: 4 (Mus musculus), SEQ ID NO: 7 (Homo sapiens) and
  • SEQ ID NO: 9 (Rattus norvegicus). Humans L100 (SEQ ID NO: 1) is localized to chromosome 3, according to the genomic data the human L100 homolog (SEQ ID NO: 6) is located on chromosome 12.
  • a first cDNA clone of this gene family could be isolated from the rat (WO 99/40225).
  • the cDNA clone obtained in this way codes for a protein which was referred to as LlOO protein.
  • the sequence of L100 from the rat is reproduced in SEQ ID NO: 10.
  • SEQ ID NO: 11 is the corresponding amino acid sequence.
  • a gene is called an IEG if it meets three conditions:
  • IEGs Based on the kinetics of mRNA accumulation, IEGs are often divided into 3 classes.
  • Class I IEGs are often not detectable in resting or non-stimulated cells.
  • the maximum mRNA concentration is reached approximately 30-60 minutes after stimulation. After about 1.5 to 2 hours, the mRNA goes off
  • Class II IEGs reach maximum mRNA concentrations 2 hours after stimulation. The basal values are reached again after about 8 hours (examples of this are Narp, c-myc, GLUT1).
  • Class 3 genes are induced very quickly, their mRNAs accumulate over many hours. These mRNAs have a long half-life (e.g. fibronectin).
  • the proteins according to the invention and the nucleic acids L100 and L100 homologs coding for the proteins can be classified as class I IEGs.
  • the L100 cDNA from mouse shows a high homology in mouse Fanconi Anemia Complementation Group A fragment in 5 'untranslated area (AF 208 119). No match is found at the protein level. 5
  • nucleic acid sequences according to the invention with the sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 8 are understood to mean, for example, allele variants which have at least 90% homology on the derived
  • allelic variants include, in particular, functional variants which can be deleted, inserted or
  • the invention thus also relates to amino acid sequences which are encoded by the group of nucleic acid sequences shown above.
  • the invention advantageously relates to amino acid sequences which are characterized by the sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 30
  • SEQ ID NO: 8 can be encoded.
  • SEQ ID NO: 6 human L100 homolog
  • the essential biological property of the proteins according to the invention also includes, for example, the membrane domains and the domains rich in cysteine, serine and glutamine and the nuclear localization domain. This property enables the special biological effect of the proteins.
  • the isolated protein and its functional variants can advantageously be isolated from the human or animal brain.
  • Another essential biological property is the ability to interact with proteins, i.e. other receptors.
  • certain amino acids can be replaced by those with similar physicochemical properties (space filling, basicity, hydrophobicity, etc.).
  • arginine residues are exchanged for lysine residues, valine residues for isoleucine residues or aspartic acid residues for glutamic acid residues.
  • one or more amino acids can also be interchanged, added or removed in their order, or several of these measures can be combined with one another.
  • derivatives include homologs of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 8, for example eukaryotic homologs, shortened sequences, single-stranded DNA or RNA of the coding and to understand the non-coding DNA sequence.
  • Homologs of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 8 have a homology of at least 80%, preferably at least 85%, particularly at the DNA level preferably of at least 90%, very particularly preferably of at least 95% over the entire DNA range specified in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 8 ,
  • homologs of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 8 are derivatives such as promoter variants.
  • the promoters which precede the specified nucleotide sequences can be changed by one or more nucleotide exchanges, by insertion (s) and / or deletion (s), but without the functionality or effectiveness of the promoters being impaired.
  • the effectiveness of the promoters can be increased by changing their sequence, or they can be completely replaced by more effective promoters, including organisms of other species.
  • the aforementioned EST sequences are sequences which have a certain homology to parts of the sequences according to the invention, but whose function is unknown.
  • Nucleotide sequence in the range from -1 to -1000 before the start codon or 0 to 2000 base pairs after the stop codon changed in this way were that the gene expression and / or the protein expression changed, is preferably increased.
  • the nucleic acid sequences according to the invention can in principle be identified and isolated from all organisms.
  • the SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 8 or its homologues from eukaryotic organisms such as Caenorhabditis elegans, Drosophila melanogaster, the zebrafish, Danio can advantageously be used rerio, or isolate 0 mammals such as the rat, mouse or human.
  • the nucleic acid sequence (s) [the plural and singular should be synonymous for registration] can preferably be isolated from Mammalia.
  • DNA hybrids are advantageously 0.1 ⁇ SSC and temperatures between approximately 20 ° C. to 45 ° C., preferably between approximately 0 30 ° C. to 45 ° C.
  • DNA: RNA hybrids the hybridization conditions are advantageously 0.1 ⁇ SSC and temperatures between approximately 30 ° C.
  • These specified temperatures for the hybridization are, for example, calculated melting temperature values for a nucleic acid with a length of approx. 100 nucleotides and a G + C content of 50% in the absence of formamide.
  • the experimental conditions for DNA hybridization are in relevant textbooks Genetics such as Sambrook et al. , "Molecular Cloning", Cold Spring Harbor Laboratory, 1989, and can be calculated according to formulas known to the person skilled in the art, for example depending on the length of the nucleic acids, the type of hybrid or the G + C- 5 content. The person skilled in the art can obtain further information on hybridization from the following textbooks: Ausubel et al.
  • nucleic acid sequences according to the invention or their fragments ⁇ c can be used to isolate a genomic sequence via homology screening under the hybridization conditions mentioned above.
  • these regulatory sequences are sequences to which inducers or repressors bind and thus regulate the expression of the nucleic acid.
  • the natural regulation of these sequences may still be present before the actual structural genes and may have been genetically modified so that the natural regulation has been switched off and the expression of the genes increased.
  • the expression cassette can also have a simpler structure, that is to say there were no additional regulation signals before the sequence SEQ ID No: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 8 or its Homologues inserted and the natural
  • the promoter with its regulation was not removed. Instead, the natural regulatory sequence is mutated in such a way that regulation no longer takes place and gene expression is increased.
  • the expression cassette can also advantageously also function one or more so-called” enhancer sequences ".
  • nucleic acids according to the invention can be found in
  • Construct other markers such as antibiotic resistance or genes complementing auxotrophies may be included for selection on the construct.
  • Advantageous regulatory sequences for the method according to the invention are, for example, in promoters such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacl ⁇ 3, T7, Contain T5, T3, gal, trc, ara, SP6, ⁇ -P R - or in the ⁇ -P promoter, which are advantageously used in gram-negative bacteria.
  • Further advantageous regulation sequences are, for example, in
  • the gram-positive promoters ay and SP02 in the yeast or fungal promoters ADC1, MF ⁇ , AC, P-60, CYC1, GAPDH, TEF, rp28, ADH.
  • the particularly advantageous Mammilia promoters such as the promoters of CMV, RSV, SV40, EF-l ⁇ , CAM kinase II, Nestin, L7, BDNF, NF, MBP, NSE, ß-globin, GFAP, j _g GAP443, tyrosine hydroxylase or kainate receptor subunit 1.
  • Artificial promoters for regulation can also be used.
  • the expression cassette is used for expression in a host
  • plasmids are, for example, in E. coli pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHSl, pHS2, pPLc236,
  • the plasmids mentioned represent a small selection of the possible plasmids.
  • Vectors which are advantageous for mammals are, for example, pcDNA3.1, pci-neo, pRc / CMV2 or pRc / RSV.
  • Other plasmids are well known to those skilled in the art and can be found, for example, in the book Cloning Vectors (Eds. 35 Pouwels P.H. et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).
  • the expression cassette for the expression of the other genes contained additionally contains 3 'and / or 5' 40 terminal regulatory sequences for increasing expression, which are selected depending on the selected host organism and gene or genes for optimal expression.
  • regulatory sequences are intended to enable targeted expression of the genes and protein expression. Depending on the host organism, this can mean, for example, that the gene first is expressed or overexpressed after induction, or that it is immediately expressed and / or overexpressed.
  • the regulatory sequences or factors can preferably have a positive influence on the gene expression of the introduced genes and thereby increase them.
  • the regulatory elements can advantageously be strengthened at the transcription level by using strong transcription signals such as promoters and / or "enhancers".
  • an increase in translation is also possible, for example, by improving the stability of the mRNA.
  • a preferred embodiment is the linkage of the nucleic acid sequence according to the invention to a promoter, the promoter coming 5 'up stream.
  • Regulation signals such as terminators, polyadenylation signals, enhancers can be used in the expression cassette.
  • the expression cassette advantageously also contains further nucleic acid sequences which code for proteins which interact with L100 or its homologues. Examples of such sequences are the sequences SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.
  • the recombinant expression cassette or the nucleic acid construct or gene construct is advantageously inserted into a host-specific vector which enables optimal expression of the genes in the host.
  • vectors are well known to the person skilled in the art and can be found, for example, in the book cloning
  • vectors include all other vectors known to the person skilled in the art, such as phages, viruses such as SV40, CMV, baculovirus, adenovirus, transposons, IS elements, phasmids,
  • cosmid linear or circular DNA. These vectors can be replicated autonomously in the host organism or can be replicated chromosomally.
  • the present nucleic acid sequence or the expression cassette can be introduced and expressed in vectors in suitable systems in a manner known to those skilled in the art.
  • the nucleic acid sequences according to the invention, their homologues, their functional equivalents or derivatives are advantageously introduced and expressed as a recombinant expression cassette or as a vector in a suitable system.
  • familiar cloning and Transfection methods used to express the nucleic acids mentioned in various expression systems. These systems are described, for example, in Current Protocols in Molecular Biology, ed. F. Ausubel et al. Wiley Interscience, New York 1997.
  • the expression cassette according to the invention or the vector containing the nucleic acids according to the invention can also advantageously be introduced into the organisms in the form of a linear DNA and integrated into the genome of the host organism via heterologous or homologous recombination.
  • This linear DNA can consist of a linearized vector such as a plasmid or only of the nucleic acid construct or the nucleic acids according to the invention.
  • prokaryotic or eukaryotic organisms are suitable as host organisms which enable expression of the nucleic acids according to the invention, their allele variants, their functional equivalents or derivatives or the recombinant nucleic acid construct.
  • Host organisms are, for example, bacteria, fungi, yeasts, plant or animal cells.
  • Preferred organisms are bacteria such as Escherichia coli, Streptomyces, Bacillus or Pseudomonas, eukaryotic microorganisms such as Saccharomyces cerevisiae, Aspergillus, higher eukaryotic cells from animals or plants, for example Sf9, HEK293 or CHO cells, eukaryotic host organisms are particularly preferred, or mammals are particularly preferred Mammalian cells such as Sf9, HEK293 or CHO cells.
  • the gene product can also be used in transgenic organisms such as transgenic animals e.g. Mice, sheep or transgenic plants are brought to expression, transgenic animals are preferred.
  • transgenic organisms can also be so-called knock-out animals or plants.
  • the recombinant prokaryotic or eukaryotic host organism according to the invention containing at least one nucleic acid sequence according to the invention or at least one expression cassette or at least one vector according to the invention.
  • the combination of the host organism and the vectors suitable for the organisms such as plasmids, viruses or phages such as, for example, plasmids with the RNA polymerase / promoter system, the phages 1, Mu or other tempered phages or transposons and / or further advantageous regulatory sequences form an expression system.
  • expression systems is preferably understood to mean, for example, the combination of mammalian cells such as CHO cells and vectors such as pcDNA3neo vector which are suitable for mammalian cells.
  • the nucleic acid sequences of the present invention from humans and mice as well as their homologues form a previously unknown gene family which strongly agree in a domain which has homology to metallothioneins and in a putative nuclear localization sequence (FIG. 1).
  • the RNA products are expressed, for example, after maximum electric shock in the brain of rats (hippocampus).
  • the functional data indicate an involvement of the gene products in neuronal cell death.
  • the expression of the genes can be activated not only by electroshock but also by further stimuli which, for example, cause stress, such as, for example, injections of kainate and / or pentylene tetrazole.
  • MT metallothionein
  • the MT I and II mice are ubiquitously expressed throughout development and are regulated by metals, glucocorticoids and inflammatory stress signals.
  • MT III is predominantly expressed neuronally.
  • MT IV is expressed in epithelial cells.
  • the MT bind primarily copper, in mammals zinc, whereby zinc can be replaced by copper and cadmium ions.
  • Cellular cadmium resistance is achieved by multiplying the MT locus.
  • MT presumably serve as chaperones for metalloproteins, as a reservoir for metals and / or prevent the toxicity of metals.
  • MT III knockout mice show increased sensitivity to kainate-induced epileptic seizures and increased neurotoxicity to kainate in certain regions of the hippocampus, whereas mice are protected by overexpression of MT-III against kainate-induced cell death [Erickson et al., ( 1997), J. Neurosci. 17: 1271-1281].
  • MT-I Overexpression of MT-I shows a protective effect in certain animal models for ischemia (van Lookeren Campagne et al., (1999), Proc. Natl. Acad. Sei. U.S.A. 96: 12870-12875).
  • Metallothioneins are mainly expressed cytosolically and develop their protective effect there.
  • the homology between the LlOO gene family and its homologues and the metallothioneins is limited to the short cysteine-rich regions, so that L100 and its homologues form an independent family and cannot be counted among the metallothioneins.
  • Homology comparisons with ESTs from the Embl database show that L100 is expressed in Caenorhabditis elegans, Drosophila melanogaster and zebrafish, Danio rerio and probably developed early in evolution.
  • the metallothioneins are about 60 amino acids in length, with about 39% of the amino acids corresponding to the L100 consensus sequence. s.
  • the L100 homologs are proteins of approximately 535 to 589 amino acids, so that L100 homologs form an independent family. At least 2 L100 homologs can be detected in humans and in the rat (Appendix 1: known ESTs for L100 homologs in the EMBL database, status March 14, 2003), which, however, have no homologies to known proteins except in the cysteine-rich domain.
  • LlOO overexpression leads to cell death.
  • IEGs are genes, the expression of which is increased very rapidly by a stimulus. In neurons they are functionally involved in learning processes, memory, synptic transmission and neuronal plasticity.
  • Advantageous nucleic acids according to the invention code, for example, for the human or murine form of L100 or for their homologues or for homologues in the rat.
  • proteins can be identified which have specific binding affinities for the protein according to the invention.
  • Nucleic acids that code for proteins that have specific binding affinities for the protein can also be identified in this way.
  • the two-hybrid system or other biochemical methods are advantageously used alone or in combination. Interaction domains of the protein according to the invention and thus pharmacotherapeutic intervention points can thus be identified.
  • the invention therefore relates to the use of the two-hybrid system or biochemical methods for identifying the interaction domains of L100 and the use for pharmacotherapeutic intervention.
  • Another particularly advantageous subject of the invention are protein complexes from the LlOO protein and at least one other protein interacting with L100, such as, for example, the protein phosphatase 1, septin, chalice or zinc finger proteins.
  • Structural analyzes of the protein according to the invention specifically allow substances to be found which have a specific binding affinity.
  • L100 makes it possible, using the two-hybrid system or other assays, to narrow down the amino acids responsible for the interaction and to find substances with which the interaction between the two proteins can be influenced.
  • An object according to the invention is therefore a method for finding substances which bind specifically to a protein with the amino acid sequence according to the invention described above or to a protein complex according to the invention, which comprises one or more of the following steps:
  • the binding is detected by measuring the antagonization or agonization of the LlOO activity or by measuring the physiological effect of L100.
  • the interaction of proteins with the amino acids according to the invention can be detected using the two-hybrid system.
  • the methods can be carried out by expressing the proteins in eukaryotic cells and linking them to a reporter assay such as, for example, the gfp protein or other fluorescence assays for the activation of the LlOO protein.
  • a reporter assay such as, for example, the gfp protein or other fluorescence assays for the activation of the LlOO protein.
  • the invention further relates to a method for the qualitative and quantitative determination of proteins with the amino acid sequence according to the invention in a biological or other sample, which comprises one or more of the following steps:
  • the LlOO ligand binding is used for detection (see example 11).
  • the protein activity or amount of the LlOO proteins or homologs can be determined via antibodies.
  • the invention therefore furthermore relates to a method for quantifying the protein activity or amount of a protein.
  • the regulatory sequences of the nucleic acids according to the invention in particular the promoter, the enhancers, the so-called locus control regions, silencers or respective partial sequences thereof can be used for the tissue-specific expression of this and / or other genes. This results in the possibility of performing neuron-specific gene expression from nucleic acid constructs.
  • cis-regulatory sequences can also be very far from the start of the transcription, it is advantageous to include very large genomic areas in the analysis.
  • vector systems with a very high cloning capacity, such as, for example, BACs or YACs (bacterial artificial chromosomes, yeast artificial chromosomes).
  • the reporter gene can be inserted into the vector via homologous recombination and its expression can be examined
  • the regulatory sequences of the nucleic acids according to the invention in particular the promoter, the enhancers, locus control regions and silencers or respective partial sequences thereof, can be used for the tissue-specific expression of sequences according to claim 1 and further nucleic acid sequences. This results in the possibility of performing neuron-specific gene expression of nucleic acids in advantageous constructs.
  • the construct with the regulatory sequences can be linked to other cDNAs in order to create animal models in which the respective cDNA is expressed region-specifically (see for example Oberdick et al., (1990), Science, 248: 223-226).
  • this can also be the expression of sequence-specific DNA recombinases such as CRE recombinase, FLP recombinase or their derivatives.
  • Antibodies are understood to mean, for example, polyclonal, monoclonal, human or humanized or recombinant antibodies or fragments thereof, single chain antibodies or synthetic antibodies.
  • Antibodies according to the invention or their fragments in principle include all immunoglobulin classes such as IgM, IgG, IgD, IgE, IgA or their subclasses such as the subclasses of IgG or their To understand mixtures.
  • IgG and its subclasses such as IgGi, IgG 2 , IgG 2a / IgG 2b , IgG 3 or IgGjj are preferred.
  • the IgG subtypes IgGi or IgG b are particularly preferred.
  • Fragments include all shortened or modified antibody fragments with one or two binding sites complementary to the antigen, such as antibody parts with a binding site formed by light and heavy chains corresponding to the antibody, such as Fv, Fab or F (ab ') fragments or single-strand fragments , called. Shortened double-stranded
  • fragments like Fv-, Fab- or F (ab '). These fragments can be obtained, for example, enzymatically by cleaving off the Fc part of the antibodies with enzymes such as papain or pepsin, by chemical oxidation or by genetic engineering manipulation of the antibody genes. Genetically modified non-recombinant fragments of the antibodies.
  • the antibodies or fragments can be used alone or in mixtures.
  • the antibody genes for the genetic engineering manipulations can be isolated in a manner known to the person skilled in the art, for example from the hybridoma cells (Ausubel et al.,
  • antibody-producing cells are attracted and the mRNA if the optical density of the cells is sufficient, for example by cell disruption with guanidinium thiocyanate, acidification with sodium acetate, extraction with phenol, chloroform / isoamyl alcohol, precipitation with isopropanol and washing with ethanol
  • the reverse transcriptase is then used to synthesize cDNA from the mRNA.
  • the synthesized cDNA can be directly or after genetic manipulation, for example by site directed mutagenesis, introduction of insertions, inversions, deletions or bases.
  • Bacterial fungal or yeast vectors such as pBR322, pUC18 / 19, pACYC184, lambda or yeast mu vectors are preferred for cloning the genes and for expression
  • the cDNA, the genomic DNA, the regulatory elements of the nucleic acid sequences according to the invention, and also the polypeptide, as well as partial fragments thereof, can be used in recombinant or non-recombinant form to prepare a test
  • This test system is suitable for measuring the activity of the promoter or the protein in the presence of the test substance. These are preferably simple measurement methods (colorimetric, luminometric, based on fluorescence or radioactive) which allow the rapid measurement of a large number of test substances (Böhm et al., (1996), "Active ingredient design.” Spektrum-Verlag, Heidelberg).
  • the test systems described allow the search of chemical or biological libraries for substances which have agonistic or antagonistic effects on proteins with the amino acid sequence according to the invention or the protein complex containing at least one protein according to the invention and at least one further protein interacting with this protein.
  • the determination of the amount, activity and distribution of the protein according to the invention or its underlying mRNA in the human body can serve for diagnosis, detection of the predisposition and for monitoring in certain diseases.
  • the nucleic acid sequence according to the invention and its genomic DNA can be used to make statements about genetic causes and predispositions of certain diseases. Both DNA / RNA samples and various types of antibodies can be used.
  • the nucleotide sequence described or parts thereof in the form of suitable samples are used to detect point mutations or deletions / insertions / rearrangements.
  • the present nucleic acid sequence according to the invention, the expression cassette or the vector and the functional equivalents, homologs or derivatives of the nucleic acids, the protein encoded by them with the amino acid sequence according to the invention or the protein heteromer ( protein complex) according to the invention with one of the proteins shown in Example 15 and reagents derived therefrom (Oligonucleotides, antibodies, peptides) can be used to diagnose and treat neurodegenerative diseases.
  • Monitoring of the treatment of diseases can also be carried out. This affects e.g. the course assessment of diseases, the assessment of therapeutic success and the grading of a disease.
  • the invention further relates to a method for the qualitative and quantitative detection of a nucleic acid according to the invention in a biological sample, which comprises the following steps:
  • the invention relates to a method for the qualitative and quantitative detection of a protein heteromer or a protein according to the invention in a biological sample, which comprises the following steps:
  • a biological sample is usually taken from a healthy organism.
  • the invention further relates to a method for finding substances which are specific to a protein having the amino acid sequence according to the invention or to a protein complex which comprises at least one protein according to the invention and at least one bind further protein which interacts with the protein according to the invention, which comprises one or more of the following steps:
  • Protein with the amino acid sequence according to the invention or on the protein complex or an effect on the receptor function are known.
  • the invention relates to a method for finding substances which bind specifically to the protein or the protein complex according to the invention and thereby cause inhibitory or activating functional effects on the LlOO signal transmission in neurons.
  • the protein can be applied, naturally or recombinantly, directly or by suitable measures in the form of its coding nucleic acid (i.e. DNA or RNA). Both viral and non-viral vehicles can be used for this.
  • Another way is to stimulate the endogenous gene in the body with suitable substances. Such substances can be found, for example, by determining their effect on the transcription elements of the LlOO gene.
  • nucleic acids according to the invention or complementary nucleic acid sequences derived therefrom can be used for the production of medicaments for the treatment of diseases which can be influenced positively by modulating the gene expression of L100.
  • Proteins, protein fragments or peptides or parts thereof according to the invention can also be used.
  • the invention also relates to the use of antibodies or antibody fragments or antibody mixtures against the protein according to the invention or against the protein heteromer for the production of medicaments for the treatment of diseases which can be positively influenced by modulating the activity or amount of L100 protein.
  • Substances which bind specifically to the protein according to the invention or the proteins according to the invention or the protein complex can be used for the production of medicaments for the treatment of diseases which can be influenced positively by modulating the activity or amount of L100 protein.
  • diseases or diseases mentioned are diseases associated with apoptosis and / or neurodegenerative diseases such as epilepsy, ischemia, dementia, Parkinson's, Huntington's, Alzheimer's or CNS trauma.
  • Diseases such as epilepsy, Alzheimer's, ischemia, stroke or CNS trauma are preferred.
  • nucleic acid sequences according to the invention can also be expressed in the form of therapeutically or diagnostically suitable fragments.
  • vector systems or oligonucleotides can advantageously be used which extend the cDNA by certain nucleotide sequences and thus code for modified polypeptides which serve for easier purification.
  • anchors so-called “tags” are known in the literature, for example, hexa-histidine anchors or epitopes that can be recognized as antigens of various antibodies (described, for example, in Harlow, E. and Lane, D., 1988, Antibodies : A Laboratory Manual, Cold Spring Harbor (NY) Press).
  • anchors can be used to attach the proteins to a solid support such as a polymeric matrix, which can be filled, for example, in a chromatography column, or to a microtiter plate or to another support.
  • these anchors can also be used to recognize the proteins.
  • conventional markers such as fluorescent dyes, enzyme markers, which form a detectable reaction product after reaction with a substrate, or a radioactive marker alone or in Combination with the anchors can be used to derivatize the proteins.
  • the nucleic acid sequences according to the invention can also be used as markers for human or animal inherited diseases or for gene therapy.
  • L100 as a potential transcription factor
  • the binding of the protein to the radio-labeled DNA leads to an increase in the molecular weight of the complex. This leads to a slower running speed of the complex compared to that of the individual DNA molecule (gel retardation) in a gel electrophoresis. After autoradiography, this leads to a shift in the band (shift) towards a higher molecular weight.
  • the MCKay assay can be used as further proof: An antibody that does not impair the DNA binding of the protein is incubated with cell nucleus extracts and the radioactive DNA and then immunoprecipitated. The proteins are removed by phenol extraction and the remaining DNA is evaluated electrophoretically. The radioactivity measured is a measure of the affinity of the protein for the DNA. Affinities of transcription factors for their target DNA are approximately 10 8 to 10 1 M -1 . If the binding site for L100 on the target DNA is to be characterized, DNA footprinting experiments are carried out. The principle of DNase I in vitro footprints is that DNA, to which a protein is bound, is protected against degradation by DNasel. Molecular analysis of transcription factors showed that they consist of a domain which is responsible for the sequence-specific recognition of the DNA and binding to the DNA, and another domain which interacts with the basic transcription machinery [Papavassiliou AG
  • Active substances can therefore modulate the activity of transcription factors, among other things, by the following mechanisms: they can prevent their degradation or the translocation of the transcription factor into the cell nucleus; specific binding to the transcription factor could prevent the interaction with other factors in the cell nucleus; binding to the promoter of regulated target genes could be prevented [Papavassiliou AG (1997), Mol. Med. 3: 799-810, Papavassiliou AG (1998), Mol. Med. Today 4: 358-366, Papavassiliou AG (2000) , J. Cancer Res. Clin. Oncol., 126: 117-118].
  • the positive modulation of neurodegenerative processes could therefore be via the Regulation of the transcriptional activity take place [Dai et al.
  • the determination of the amount, activity and distribution of the protein according to the invention or its underlying mRNA in the human body can serve for diagnosis, predisposition and monitoring for certain diseases.
  • the sequence of the cDNA and the genomic sequence can be used to make statements about the genetic causes and predispositions of certain diseases.
  • both DNA / RNA samples and unnatural DNA / RNA samples as well as antibodies of various types can be used.
  • the nucleotide sequence described or parts thereof in the form of suitable samples are used to detect point mutations or deletions / insertions.
  • Example 1 Screening method for determining the human L100 sequence SEQ ID NO 1
  • SEQ ID NO 1 was determined by repeatedly searching a human hippocampal bank (oligodT and randomly primed) in Lambda ZAP (Stratagene). A cDNA (2.97 kb length) L100 from rats (SEQ ID NO: 10) after radioactive labeling was used as a probe (random labeled label kit from Röche Diagnostics). The hybridization was carried out in 5xSSC, 5% Denhardt's solution, 0.025% sodium pyrophosphate, 0.1 mg / ml yeast tRNA and 50% formamide solution at 40 ° C overnight. Was washed with 2xSSC, 0.1% SDS solution at 60 ° C.
  • Example 2 Screening method for determining the genomic sequence of L100 in the mouse
  • the 2.97 kb rat LlOO fragment (SEQ ID NO: 10) fragment was radioactively labeled and used to hybridize a mouse genomic cosmid library (129 / Ola, RZPD, Berlin). Cosmid DNA was isolated from a positive clone. This clone was verified as LlOO positive by means of various restriction digests and hybridizations with L100 probes (by comparing the band patterns obtained with those of genomic DNA from mice). Various fragments from the cosmid were subcloned into a plasmid vector and sequenced using a transposon insertion method (GPS-1, New England Biolabs, Beverly, MA; USA), and the sequences were assembled using the SeqMan program (Lasergene, Madison, WI, USA) ,
  • the genomic sequence of L100 of the mouse is shown in SEQ ID NO: 5.
  • the cDNA of L100 of the mouse, which was created on the basis of homology to the rat, is shown in SEQ ID NO: 3.
  • the comparison of the mouse genomic sequence and the cDNA sequence of the rat shows that the coding region for the L100 protein in the mouse is interrupted by introns.
  • the exact exon / intron structure is shown in Figure 16.
  • Example 3 Screening method for determining the sequence of the homologue of L100 in humans
  • SEQ ID NO: 1 BLAST [BALSTN 2.0.11, Altschul et al., (1997), Nucleic. Acid Research., 25: 3389-3402] related ESTs in rat and human. Primers were derived from the ESTs AI205148 and AA305194, probes from the hippocampus cDNA (human) were amplified, radioactively labeled and the human hippocampus bank (oligodT and randomly primed, Stratagene) used in Example 1 was searched under the conditions specified in Example 1. A positive lambda clone contained the open reading frame for SEQ ID NO: 6.
  • Example 4 Screening method for determining the sequence of the homologue of L100 in the rat
  • LlOO-specific sense and antisense cRNA samples were produced from this template using T3 RNA polymerase and T7 RNA polymerase, digoxigenin-labeled UTP and standard NTPs.
  • Frozen rat brains and embryos were cut into 15 ⁇ m slices in a cryostat (Leica GmBH) at -20 ° C, fixed with 4% paraformaldehyde in PBS, permeabilized in 0.2% Triton-X 100 and with antisense and sense LlOO cRNA samples at 65 ° C overnight in 50% formamide, 5X SSC hybridized. These sections were washed with 0.2XSSC at different temperatures and the dioxigenin label was detected with a specific antibody (Kuner et al., (1999),
  • the sense samples gave no signals, so that specific LlOO signals can be assumed for the antisense signals.
  • the rat LlOO mRNA is ubiquitous and weakly expressed in the rat embryo and strongly expressed in the liver, lungs, brain, retina and in peripheral neurons. (Figure 3).
  • the mRNA of rats LlOO is strongly expressed in the hippocampus, cerebellum, olfactory bulb, striatum, cortex and the amygdala during the postnatal development of the brain (days 4 and 7). In the adult animal, the expression of LlOO is down-regulated when the development of the brain is complete.
  • LlOO mRNA is expressed in very small amounts in adult brain neurons and cannot be detected in astrocytes.
  • the fragment from SEQ ID NO: 1 was radioactively labeled with a- 32 P-dCTP using the random primed labeling kit (Boehringer Mannheim).
  • a Northern blot (10 ⁇ g total RNA from humans: brain, heart, skeletal muscle, large intestine, thymus, spleen, kidney, liver, small intestine, placenta, lungs and peripheral blood leukocytes) in QuickHyb solution (Stratagene ) hybridized for one hour at 68 ° C and then washed at 60 ° C with 0. lxSSC solution.
  • Example 7 Expression of LlOO in the rat brain after seizures
  • Kainate injections are a recognized animal model for frontal lobe epilepsy. 1.5 hours after intraperitoneal injection of kainate (10 mg / kg in PBS i.p.), LlOO was massively upregulated in the cerebellum, hippocampus, cortex and thalamus. Maximum upregulation in cerebellar granule cells and some neurons of the entorhinal, frontal and orbital cortex and hippocampus could be observed up to 6 hours after the Kainat injection (FIG. 4). Normal values were reached again 24 hours after the injection (FIG. 4).
  • Zinc is stored in glutamatergenic, synaptic vesicles in the brain, released during synaptic activation and taken up by post-synaptic neurons in the cortex and hippocampus. Accumulation of zinc after kainate injections in some external neuronal populations
  • the so-called kindling model is an accepted animal model for epileptogenesis.
  • stage 2 convulsions SS2; facial clonus and / or involuntary nodding of the head
  • SS5 stage 5 convulsions
  • RNA was pooled from 4 animals per experimental group. 3 RNA pools were used for the further investigation. Five micrograms of total hippocampal RNA were transcribed in a reverse transcription with a Tis (G / A / ON primer using Superscript II (Life Technologies) according to the manufacturer's protocols into first-strand cDNA. To measure the relative amount of LlOO mRNA in Quantitative PCR reactions with a LightCycler (Röche) were carried out on the cDNAs, a PCR method in capillaries, in which the online detection of the intercalated SYBR Green Fluorescent dye, the amplification of the DNA can be measured and quantitative statements about the number of template molecules can be made.
  • a PCR method in capillaries in which the online detection of the intercalated SYBR Green Fluorescent dye, the amplification of the DNA can be measured and quantitative statements about the number of template molecules can be made.
  • the amplified products were detected with SYBR Green (Molecular Probes).
  • the PCR reactions were carried out according to the instructions in the protocols of the manufacturer of the DNA Master SYBR Green (Röche).
  • For the amplification of LlOO 60 cycles with 65 ° C annealing temperature and a final concentration of 5mM MgCl 2 were carried out using the following primers:
  • Primer 1 5 '-GACCATCGGTGATCAAAACTC-3'
  • Primer 2 5 '-ACATCAGCTAATGAAGGGCATA-3'
  • Primer 1 5 '-AAGTTTGTCATTCGGAACATTGT-3'
  • LlOO is a marker for overstimulated neurons, which can be derived from the kainate injections and the increased expression level of LlOO in the kindling model, one would also expect an upregulation of LlOO after glutamate receptor activation. Therefore, quantitative RT-PCR studies with differentiated, cortical neurons were carried out after 14 days in vitro cultivation. Glutamate was applied to the neurons in a concentration of 100 ⁇ M in HSS for 5 minutes, causing excitatory cell death after 18-24 hours. Control cultures were treated with HSS alone. All cultures were then washed and the conditioned medium was changed. The RNA from the neurons was isolated 6 hours after glutamate administration using RNeasy columns from Quiagen.
  • the immediate early genes Fos and Jun are already known to be important downstream mediators of long-term effects after glutamate activation. Therefore, LlOO may also play an important role in pathophysiological cascades of glutamate-like activation.
  • Example 6 The conditions chosen in Example 6 were also used for the following experiments.
  • SEQ ID NO: 8 as a probe was hybridized with a Northern blot (10 ⁇ g total RNA from the hippocampus of rats or control animals subjected to multiple electric shocks without electroshock treatment) for one hour at 68 ° C. and at 60 ° C. with 0. lxSSC solution washed. After the screen had been placed on the screen for 24 hours, the Fuji Phosphoimager FLA2000 was able to detect a 4-fold increase in mRNA in the hippocampus after the animals were killed 4 hours after the last electric shock (FIG. 6). On a blot with 10 ⁇ g Brain-specific expression of SEQ ID NO: 8 was detected in total RNA from the rat brain, liver, heart, kidney and testis.
  • the mRNA for SEQ ID NO: 8 was detected with the aid of the in situ hybridization described above in the adult rat brain and is evenly distributed in the brain, stronger signals for the SEQ ID NO 8: mRNA being present in the cerebellum, cortex and hippocampus are. In the hippocampus and dentate gyrus, SEQ ID NO: 8 is expressed especially in CA3 and CA4 regions, not in the CAl section. After 4xMECS treatment, a moderate upregulation of SEQ ID NO: 8 can be seen in the cerebellum and cortex (FIG. 7). A strong signal has now been observed in the CA sector of the hippocampus. No change in gene expression occurred in rats after kainate or PTZ treatment.
  • LlOO and LlOO homologues may not appear to be essential for the functioning of the adult rat brain in the normal, physiological state, but they play an important role in the development of the brain and in pathological conditions associated with massive neuronal overexcitation of the brain, have an essential meaning.
  • N-terminal GST-L100 fusion protein was achieved by cloning the open reading frame of SEQ ID NO: 10 without the first methionine from LlOO into the pGEX-6P vector from.
  • the recombinant protein was induced in protease-free bacteria (SG200.43 + pDMI.l) by adding 200 ⁇ M IPTG for 3 hours, the bacterial pellet was obtained by centrifugation at 3000xg for 20 min and then in PBS and 0.5% EDTA with the addition of a proteinase inhibitor Cocktails from Röche Diagnostics sonified and then pelleted again at 6000xg for 10 min.
  • the supernatant was centrifuged again with 10000xg and the L100-GST or GST protein extracts were affinity-purified on the glutathione-Sepharose column from Pharmacia Biotech. Elution was carried out with 10 mM glutathione in 50 mM Tris-HCl under metal-free conditions.
  • the protein content was determined using the BCA assay and the degree of purity was determined using Coomassie-stained polyacrylamide gels.
  • the zinc content of recombinant L100-GST or GST was determined using N- (6-methoxy-8-quinolyl) -p-toluene sulfonamide (TSQ, Molecular Probes).
  • TSQ only fluoresces when zinc is complex bound and can therefore be used for the quantitative detection of zinc. Same Concentrations of L100-GST or GST were spotted on glass slides and treated with 0.05% solution of TSQ (Molecular Probes) in acetone, air dried and analyzed under a fluorescence microscope with an excitation of 330 nm and an extinction of 5 385 nm. TSQ alone gave no fluorescent signal, GST protein a weak signal and GST-L100 a significantly stronger signal. The weak signals from GST could be contaminations with metal-binding proteins from E. coli, the strong signal from GST-L100 showing that LlOO is formed in E. coli as a zinc-complexing protein (FIG. 9).
  • HEK293 cells were incubated in standard MEM medium (Gibco BRL), 10% fetal calf serum (Sigma Immunochemicals) at 37 ° C., 5% CO and 95% atmospheric humidity and transfected with LlOO plasmids according to the calcium phosphate method.
  • the LlOO protein accumulated in the cytoplasm of the transfected HEK293 cells and was evident in the cell protrusions that resembled apoptotic bodies. At the same time, condensation of the chromate scaffold was observed in the LlOO transfected cells, which then died. For the pro-apoptotic phenotype, it did not matter whether LlOO was tagged or not. As a negative control, cells were transfected with the empty vector or control proteins (GBR2-Flag, Kuner et al., (1999), Science, 283, 74-77) (FIG. 11).
  • LlOO LlOO in the brain
  • primary neurons were transfected with the labeled LlOO sequence. These neurons were obtained from rat hippocampi embryonic day 18-19, dissociated and coated on plates coated with poly-L-lysine (Sigma Chemicals, 0.1%) and laminin (Sigma chemicals, 1-2 ⁇ g / cm 2 ) in a medium containing 1% horse serum , Contains 3% glucose, standard additives and hormones, cultivated at 37 ° C, 5% C0 and 95% relative humidity. After one day in vitro, the neurons continue to develop and 8 to 9 days later they develop spines that contain synapses.
  • the neurons were cultivated in vitro for 4 days and then transfected with Myc-LlOO-DNA or control DNA, which was complexed with the lipophilic agent Effectine (Qiagen GmBH).
  • the cells were then fixed, permeabilized and stained with a monoclonal antibody against the Myc epitope (Invitrogen, 4 ⁇ g / ml and an anti-mouse FITC conjugated second antibody (Jackson Immunoresearch Labs, 7.5 ⁇ g / ml) as described above.
  • 15 hours after Transfection was mostly localized in the cell nucleus and partly in the cytoplasm of the neurons ( Figure 12). After 18 hours, LlOO was mainly detected in the cytoplasm and the neurites.
  • HEK293 cells were transfected with LlOO or control plasmid and counterstained with flous-conjugated annexin (Boehringer Mannheim), a dye that stains phosphatidylserines that are part of the cell wall.
  • flous-conjugated annexin Boehringer Mannheim
  • apoptotic cells expose the phosphatidylserines and are stained with flous annexin.
  • the apoptotic cells were included
  • LlOO cells were mostly propidium iodide negative and showed a stronger staining with flous annexin ( Figure 14). The negative controls again demonstrate induction of apoptosis by heterologous overexpression of LlOO in cells.
  • Nuclear staining was carried out with maximum dye (33342, Molecular Probes) and showed condensation of the DNA 18 hours after LlOO transfection, a sign of cell degeneration (FIG. 15). In comparison to the control vector, cell death was triggered by LlOO, which was detected by the so-called TUNEL staining. The method detects the free 3 -OH groups after fragmentation of the DNA.
  • the neurons were transfected with Clontech's LlOO-Myc-Tag expression plasmid or EYFP expression vector as described above.
  • the cells were washed with PBS and preincubated with TUNEL dilution buffer (Röche Diagnostics, in 30 mM Tris-HCl, 140 mM Sodium cocodylate and ImM Cobalt chloride) and further incubated with terminal deoxynucleotidyl transferase (Röche Diagnostics) with biotinylated dNTPs in the TUNEL dilution buffer according to the manufacturer's instructions for 60 min at 37 ° C.
  • TUNEL dilution buffer Rosha Diagnostics, in 30 mM Tris-HCl, 140 mM Sodium cocodylate and ImM Cobalt chloride
  • the cells were washed three times with PBS for 5 min, blocked with 10% NGS for 15 min, incubated with TRITC-streptavidin (Jackson Immunoresearch Labs) for 25 min, counterstained with Hoechst 33342, washed 2 5 min with PBS and sealed in aqueous
  • the targeted mutation of the LlOO gene in the germ line of the mouse and the analysis of the resulting phenotype can provide important additional information about the (patho) physiological mechanisms in which the LlOO gene is involved.
  • a so-called knock-out mouse ie a mouse without a functional LlOO protein
  • a so-called targeting construct was first produced. Two genomic fragments flanking the coding region of LlOO (corresponding to positions 2866 to 4083 and 9813 to 13500 in the sequence SEQ ID NO: 5 according to the invention) were used as homology arms for homologous recombination in embryonic stem cells (ES) in the vector pHM2 (Kaestner et al., (1994), Gene, 148, 67-70).
  • This vector carries a neomycin resistance cassette and allows a reporter gene to be inserted into the allele to be mutated.
  • the lacZ reporter gene of the vector was fused to the 5 'untranslated region of LlOO and was therefore under the control of the endogenous LlOO promoter.
  • the lacZ cassette now completely replaces the open reading grid from LlOO ( Figure 16).
  • the DNA should be electroporated into embryonic stem cells and G418-resistant clones selected. Genomic DNA is to be isolated from these clones and examined for so-called nested PCR for homologous recombination between targeting construct and endogenous LlOO allele.
  • 16 shows the genomic structure of LlOO and the chosen homology arms for the homologous recombination, as well as the primers for the PCR detection of the recombination.
  • genomic DNA can be used as a template to determine whether the recombination has taken place in the ES cells.
  • As a positive control you can use this in genomic DNA Spike in plasmids and thus determine the sensitivity of the PCR method.
  • LlOO codes for a protein of 581 amino acids.
  • the L100 protein has a cysteine-rich domain (235-273) which is 33% identical to metallothioneins, a potential coiled-coil domain (119-155), as well as a serine-rich domain (18-42) and one Glutamate-rich (402-407) region.
  • a potential core localization signal 200-207 is detectable, which could be responsible for the transport of LlOO into the core (SEQ ID NO: 10) ( Figure 1).
  • the cDNA coding for the carboxy terminus of the LlOO gene (SEQ ID NO 11, amino acids 407-581) was prepared with the specific LlOO primers L100-5 '-Y2H-407 (ACAGCGACGTCGACG-GAGGGAGTGTG-GGCAACTT) and L100-3' Y2H-581 (ATAGTTTAGCGGCCGCTT-ACACAGGCA-CAGGGTC) from the LlOO rat cDNA amplified in a PCR (polymerase chain reaction), subjected to a restriction with the enzymes Notl and Sall and then cloned into the Notl / Sall pre-cut vector pDBleu (Gibco).
  • the resulting construct (L100-407-581) codes for a protein in which the Gal4 binding domain is fused to the C-terminus of the LlOO gene.
  • the cDNA coding for the amino terminus of the LlOO gene (SEQ ID NO 11, amino acids 2-315) was analyzed with the specific LlOO primers LlOO 5'-2-Y2H (CAAACGCGTCGACCGCTGGGATAC-AGAAGAAG) and L100-3'-315- Y2H (ATAGTTTAGCGGCCGCTTAGTCCTCCATGGG-GGACTC) from the LlOO rat cDNA was amplified in a PCR, and also cloned into the vector pDBleu (Gibco) after restriction digestion with the enzymes Notl and Sall. In this construct (L100-2-315) the Gal 4 binding domain was fused to the N-terminus of LlOO.
  • the constructs L100-407-581 and L100-2-315 were transformed into the yeast strain Y190.
  • HIS3 codes for the imidazoles glycerol phosphates
  • Dehydratase an enzyme in histidine biosynthesis.
  • weak protein-protein interactions can be determined from the threshold value for 3-AT resistance.
  • the yeast strains were transformed with a rat hippocampus cDNA bank MECS-induced rat, which is cloned into the vector pPC86 (Gibco). 4 * 10 6 transformants were plated on tryptophan / leucine / histidine-deficient medium with an appropriate 3-AT concentration (20mM 3-AT). After 3, 4 and 5 days of growth at 30 ° C., colonies were separated and subjected to XGAL staining. A total of 19 colonies were positive for the LlOO-N terminus and 6 colonies for the LlOO-COOH terminus as His3 and lacZ.
  • the pPC86 plasmid DNA was purified from the positive yeast colonies and transformed into the E. Coli strain Xllblue for amplification. The pPC86 DNA of the positive colonies obtained was co-transformed into the yeast strain Y190 using various pDBleu constructs.
  • the positive cDNAs from the vector pPC86 were amplified with the vector-specific primers pPC86a (GTATAACGCGTTTGGAATCAC) and pPC86b (GTAAATTTCTGGCAAGGTAGAC) and the PCR fragment obtained was sequenced.
  • PP1 is ubiquitously expressed, involved in numerous metabolic processes such as muscle contractions, cell cycle, neurotransmission (Ohkura et al., (1989), Cell 57: 997-1007; Kitamura et al., (1991), J. Biochem., 109: 307 - 310; Sasaki et al., Jpn. J. Cancer Res., (1990), 81: 1272-1280).
  • There are 2 main isoforms, the PPI alpha and delta which have almost identical catalytic domains and differ only in their NH and COOH terms (Andreassen et al., (1998), J.
  • PPl is enriched in the dendrites and plays a role in the activity of the AMPA channels, in the dopamine regulation of the Ca2 + current and in the neuropeptide regulation of the K + current [Smith FD. Et al., (1999), J. Biol. Chem. 274, 28, 19894-19900; Yan et al., (1999) Nature Neuroscience, vol. 2, nol; Allen PB. Et al., (1997) Proc. Natl. Acad. Be. USA 94, 9956-9961, Terry-Lorenzo et al., (2000), J. Biol. Chem., 275: 2439-2446; Strack et al., (1999) J.
  • SEQ ID NO: 12 shows the sequence of the protein phosphatase 1 alpha.
  • the sequence of another binding partner is hitherto unknown, it contains a 55% amino acid sequence identity to "Kelch” proteins (Xue et al., 1993 Cell, 72 (5): 681-693) and a 33% identity to the mouse zinc finger Protein 151 (Jordan-Sciutto et al., (1999), J. Biol. Chem. 274: 35262-35268; Schulz et al., 1995, BIOCHEM. J. 311: 219-224).
  • the goblet repeats consisting of 50 amino acids have the ability to bind actin (Field et al., 1999, Trends Cell.
  • Chalice proteins can have a POZ domain in the N-terminus, which is involved, among other things, in chromatin folding and the organization of the cytoskeleton in brain cells (Ahmad et al., 1998, Proc. Natl. Acad. Sci. USA, 95 (21): 12123-12128).
  • the identified sequence has 67.4% nucleotide sequence identity in 350 nt to Mayven, an actin-binding protein that is predominantly expressed in primary neurons of the hippocampus.
  • Mayven contains a BTB / POZ domain and goblet repeats and is colocalized with actin filaments in "stress fibers" and cortical actin-rich regions of the cell boundaries (cell margins) (Soltysik-Espanola et al., 1999, Mol. Biol. Cell., 10 (7): 2361-2375).
  • the sequence of the identified LlOO interaction partner with Kelch repeat is reproduced in sequence SEQ ID NO: 13. Sequence comparison to Chalice Drosophila melanogaster Ring canal protein (Chalice protein)
  • PAHMGKAFKVMNELRSKRLLCDVMIVAEDVEVEAHRWLAACSPYFCAMFTGDMSESKAK 439 P H + + + N + R LLCD V + V + + AH + VLAACS YF + F + + PQHSQRVLEQLNQQRQLGLLCDCTFVVDLVAFQHQGLLCDCTFVVDLVAFQQQQLLCLCTFVVDLVAFVQKQ
  • the term is unknown and no homology to protein binding motifs has been demonstrated.
  • This gene shows 95% sequence identity (605 bp) to Septin 6 (Kinoshita M., "Identification of mouse Septin ⁇ gene and its product", the sequence is in the EMBL database, the paper has not yet been published). Septin 6 is said to belong to the Septine family. These are GTPases that can form plasma membrane-associated filaments. Septin complexes can play an important role in vesicle transport and in the organization of proteins on the plasma membrane of neurons [Hsu et al., (1998) Neuron 20: 1111-1122].
  • Septins are involved in cytokinesis and the organization of the cytoskeleton, among others, and occur in neurofibrillary tangles in Alzheimer's patients [Kinoshita et al., (1998), Am. J. Pathol. 153: 1551-1560].
  • SEQ ID NO: 14 shows the nucleic acid sequence of Septin 6. [Fung et al. , (1999), FEBS Lett. 451: 203-208; Kinoshita et al., (1998), Am. J. Pathol.

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Abstract

La présente invention concerne de nouvelles protéines à expression spécifique, ainsi que des séquences d'acides nucléiques ou des constructions d'acides nucléiques de recombinaison, qui codent ces protéines. La présente invention concerne également des organismes hôtes ou des animaux transgéniques, qui comprennent lesdites séquences d'acides nucléiques ou lesdites constructions d'acides nucléiques de recombinaison, ainsi que des anticorps monoclonaux ou polyclonaux, qui sont dirigés contre les protéines isolées. La présente invention concerne également un procédé pour trouver des substances présentant une affinité de liaison spécifique aux protéines selon cette invention, ainsi qu'un procédé pour identifier de manière qualitative ou quantitative les séquences d'acides nucléiques selon cette invention ou les protéines selon cette invention. En outre, cette invention concerne l'utilisation desdites séquences d'acides nucléiques et desdites protéines.
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US20050147967A1 (en) 2005-07-07
DE10019901A1 (de) 2001-10-25
WO2001081623A3 (fr) 2002-11-28
AU2001254810A1 (en) 2001-11-07

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