WO2002022681A2 - Neuronal exprimiertes protein und seine verwendung - Google Patents
Neuronal exprimiertes protein und seine verwendung Download PDFInfo
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- WO2002022681A2 WO2002022681A2 PCT/EP2001/010689 EP0110689W WO0222681A2 WO 2002022681 A2 WO2002022681 A2 WO 2002022681A2 EP 0110689 W EP0110689 W EP 0110689W WO 0222681 A2 WO0222681 A2 WO 0222681A2
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Definitions
- the present invention relates to new specifically expressed proteins, nucleic acid sequences, recombinant nucleic acid constructs containing the nucleic acid sequences or vectors containing the nucleic acid sequences or the recombinant nucleic acid constructs which code for the proteins.
- the invention also relates to transgenic organisms containing the nucleic acid sequences, the recombinant nucleic acid constructs or the above-mentioned vectors and mono- or polyclonal antibodies which are directed against the isolated proteins.
- the invention further relates to a method for finding substances which bind with specific binding affinity to 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 occurs between 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 thus leads (eg seizure) for direct changes to important second messenger systems.
- An external stimulus thus leads (eg seizure) for direct changes to 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].
- IEGs appear to represent important intracellular regulatory points. Immediate Early Genes are functionally involved in learning processes, memory, synptic transmission and neuronal plasticity in neurons. 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 or SEQ ID NO: 5,
- 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 (Rattus norvegicus), SEQ ID NO: 3 (Homo sapiens) and SEQ ID NO: 6 (Mus museculus). These sequences are referred to below as A013 and their homologues.
- the first ESTs of the gene family according to the invention were isolated from the rat (WO 99/40225). Starting from the nucleic acid sequences SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 according to the invention, a blast search was carried out [BLASTN 2.0.11 (Jan-20-2000), Altschul et al., (1997), Nucleic Acid Res , , 25: 3389-3402] in various nucleic acid sequence banks according to EST sequences. The following sequences were found in the databases (EMBL sequences):
- 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.
- IEG immediate early genes
- 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 hardly detectable in resting or non-stimulated cells.
- the maximum mRNA concentration is reached approximately 30 to 60 minutes after stimulation. After about 1.5 to 2 hours the mRNA concentration returns to the basal values (examples are c-fos, c-jun, zif268).
- 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 RNAs accumulate over many hours. These mRNAs have a long half-life (e.g. fibronectin).
- the proteins according to the invention and the nucleic acids of A013 coding for the proteins and their homologs can be classified as class I IEGs.
- the isolated proteins according to the invention are to be understood as proteins which have an amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 or a sequence obtainable therefrom by substitution, inversion, insertion or deletion of one or more amino acid residues included, at least one of the essential biological properties Shafts of the protein shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 are retained.
- certain amino acids can be replaced by those with similar physico-chemical 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.
- proteins modified in this way with respect to SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 have at least 60%, preferably at least 70% and particularly preferably at least 90% sequence identity to the sequences in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 calculated according to the Altschul et al. [1990, J. Mol. Biol., 215: 403-410, BLAST program (version BLASTP 2. Oal9-WashU; 05-Feb-1998) over the entire length of the protein sequences].
- Functional equivalents of the sequences mentioned are to be understood as nucleic acids which code for proteins which have the biological activity of A013 and at least 50% of the activity of the sequences mentioned under SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 exhibit. These functional equivalents advantageously interact with other proteins with which they form so-called protein complexes (protein heteromers).
- An essential biological property of the proteins according to the invention is the ability to interact with Skpl via the F-box domain and the ability to bind target proteins via the substrate binding domain, which proteins are to be degraded by the ubiquitin-dependent protein degradation.
- Typical substrate binding domains of F-box proteins are so-called Leucine rieh repeats and WD domains, they are found in more than 50% of the known F-box proteins as interaction domains.
- leucine zipper domains, ring finger domains, helix-loop-helix domains, SH2 domains as well as a cyclin box act as a cyclin F Substrate binding domain (Cenciarelli et al., 1999, Curr. Biol., 9: 1177-1179; Winston et al., 1999, Curr. Biol., 9: 1180-1182).
- the essential biological properties also advantageously include the specific binding of synthetic or natural agonists and antagonists to the proteins according to the invention with the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 or their functional equivalents, their biological properties are changed or modulated.
- the proteins according to the invention and its functional variants can advantageously be isolated from the brain, the placenta, the kidney, the lungs or the heart of malia such as Homo sapiens, Mus musculus, or Rattus norvegicus.
- the invention further relates to nucleic acid sequences which code for the proteins described above, in particular for those having the primary structure shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6.
- the nucleic acid sequence from Rattus norvegicus is shown in SEQ ID NO: 1 and from Homo sapiens in SEQ ID NO: 3 and from Mus musculus in SEQ ID NO: 5.
- Allelic variants include, in particular, those functional variants which can be obtained by deleting, inserting or substituting nucleotides from the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 5, with at least one of the essential biological properties being retained remains.
- SEQ ID No: 5 is an incomplete sequence that contains only one exon (1).
- Homologous or sequence-related nucleic acid sequences can be isolated from all mammalian species including humans by conventional methods, for example by means of hoology screening by hybridization with a sample of the nucleic acid sequences according to the invention or parts thereof.
- Functional equivalents are also to be understood as homologs of SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 5, for example their homologues from other mammals, shortened sequences, single-stranded DNA or RNA of the coding and non-coding DNA sequence.
- Homologs of SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 5 have a homology on the DNA level of at least 80%, preferably at least 85%, particularly preferably at least 90%, very particularly preferably at least 95% the entire range specified in SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 5.
- Homologs of the sequences SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 5 are also to be understood as derivatives such as promoter variants.
- the promoters which precede the specified nucleotide sequences together or individually 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 or decreased by changing their sequence, or they can also be completely replaced by other promoters, including organisms of other species.
- Derivatives are also advantageously to be understood as variants whose nucleotide sequence in the range -1 to -10000 before the start codon or in other regulatory cis-flanking elements has been changed such that the gene expression and / or the protein expression is changed, preferably increased. Derivatives are also to be understood as variants that were changed at the 3 'end.
- Such functional equivalents can be isolated from other vertebrates such as Mammalia, starting from the DNA sequences described in SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 5 or parts of these sequences, for example using conventional hybridization methods or the PCR technique , These DNA sequences hybridize under standard conditions with the sequences according to the invention. Short oligonucleotides of conserved regions, for example of the F-box domain or the C-terminus, are advantageously used for the hybridization. However, longer fragments of the nucleic acids according to the invention or the complete sequences can also be used for the hybridization.
- DNA hybrids are approx. 10 ° C lower than those of DNA: RNA hybrids of the same length.
- DNA hybrids are advantageously 0.1 ⁇ SSC and temperatures between approximately 20 ° C. to 45 ° C., preferably between approximately 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 of genetics such as Sambrook et al. [(eds.), 1989. Molecular cloning: A Laboratory Manual. CSH Laboratory Press, New York] and can be calculated according to formulas known to those skilled in the art, for example depending on the length of the nucleic acids, the type of hybrid or the G + C content.
- nucleic acid sequences according to the invention or their fragments can also be used to isolate genomic sequences via homology screening using the hybridization conditions mentioned above.
- the nucleic acid sequences according to the invention are usually functionally linked to genetic regulatory elements such as transcription and translation signals. Depending on the desired application, this linkage can lead to an increase or decrease in gene expression.
- the desired host organisms are then transformed with the recombinant expression cassettes according to the invention produced in this way.
- natural regulatory elements of these sequences which lie in front of the actual structural genes, may still be present and may have been genetically modified so that natural regulation is switched off and the expression of the genes is increased.
- the gene construct can, however, also have a simpler structure, ie no additional regulation signals are inserted in front of the coding sequences and the natural promoter with its regulation is not removed. Instead, the natural regulatory sequence is mutated in such a way that regulation no longer takes place and gene expression is increased. Additional advantageous regulatory elements can also be inserted at the 3 'end of the nucleic acid sequences.
- the nucleic acid sequences for the sequences SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 5 can be contained in one or more copies in the gene 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, laclq, T7, T5, T3 , gal-, trc-, ara-, SP6-, 1-PR- or in the 1-PL-Pro ⁇ rtotor contained, which are advantageously used in gram-negative bacteria.
- Further advantageous regulatory sequences can be found, for example, in the promoters of gram-positive bacteria such as amy and SP02, in the yeast promoters such as ADC1, MFa, AC, P-60, CYCl, GAPDH TEF, rp28, ADH.
- promoters such as the promoters of CMV, RSV, SV40, EF-la, CAM kinase II, Nestin, BDNF, NGF, MBP, NSE, ß-globin, GFAP, tyrosine hydroxylase or kainate -Receptor subunit 1.
- all natural promoters with their regulatory sequences like those mentioned above can be used.
- synthetic promoters can also be used advantageously.
- regulatory sequences are intended to enable the targeted expression of the nucleic acid sequences and the protein expression. Depending on the host organism, this can mean, for example, that the gene is only expressed or overexpressed after induction, or that it is expressed and / or overexpressed immediately.
- the regulatory sequences or factors can preferably have a positive influence on the expression and thereby increase it.
- 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.
- Enhancers are understood to mean, for example, DNA sequences which bring about increased expression via an improved interaction between RNA polymerase and DNA.
- the locus control regions, silencers or respective partial sequences thereof may be mentioned as further regulatory sequences. These sequences can be used advantageously for tissue-specific expression.
- a preferred embodiment is the linkage of the nucleic acid sequence according to the invention to a promoter, the promoter coming 5 'up stream.
- a promoter can be a natural or a synthetic promoter.
- the natural promoter present in front of the nucleic acid sequences according to the invention can also be present in addition to the inserted promoters or can be replaced by these new promoters.
- a targeted genetic modification of the promoter naturally present before the sequencing is also advantageous. Further regulation signals such as 3 'terminators or polyadenylation signals or enhancers can be used functionally in the nucleic acid construct.
- expression cassette or the recombinant nucleic acid construct or gene construct also means complete vector constructs. These vector constructs or vectors are used for expression in a suitable host organism.
- the nucleic acids according to the invention are inserted into a host-specific vector which enables optimal expression of the genes in the selected host.
- Vectors are well known to the person skilled in the art and can, for example, Pouwels et al. [Cloning Vectors: Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0444904018].
- vectors also include all other vectors known to those skilled in the art, such as phages, viruses such as SV40, CMV, baculovirus, adenovirus, transposons, IS elements, phasmids, phagemids, cosmids, BACs, YACs, mammalian (mini) chro or - to understand somen vectors, linear or circular DNA.
- phages viruses such as SV40, CMV, baculovirus, adenovirus, transposons, IS elements, phasmids, phagemids, cosmids, BACs, YACs, mammalian (mini) chro or - to understand somen vectors, linear or circular DNA.
- phages viruses such as SV40, CMV, baculovirus, adenovirus, transposons, IS elements, phasmids, phagemids, cosmids, BACs, YACs, mammalian (mini) chro
- the expression of the nucleic acid sequences according to the invention or of the recombinant nucleic acid construct can advantageously be increased by increasing the number of gene copies and / or by increasing regulatory factors which have a positive influence on gene expression.
- regulatory elements can preferably be amplified at the transcription level by using stronger transcription signals such as promoters and enhancers.
- an increase in translation is also possible, for example, by improving the stability of the mRNA, or increasing the reading efficiency of this mRNA on the ribosomes, for example by changing the ribosome binding site in the 3 ′ region before the nucleic acid sequences according to the invention.
- nucleic acid sequences or homologous genes can be incorporated, for example, into a nucleic acid fragment or into a vector which preferably contains the regulatory gene sequences assigned to the respective genes or promoter activity having an analogous effect.
- those regulatory sequences are used which increase gene expression.
- nucleic acid construct according to the invention or the nucleic acids according to the invention can also be expressed in the form of therapeutically or diagnostically suitable fragments.
- vector systems or oligonucleotides can be used which extend the nucleic acids or the nucleic acid construct by certain nucleotide sequences and thus code for modified polypeptides which are used for easier purification.
- tags are known in the literature, for example, hexa-histidine anchors or epitopes that can be recognized as antigens of various antibodies (Studier et al., 1990 and Ausubel et al. [(Eds), 1998, Current Protocols in Molecular Biology, John Wiley & Sons, New York].
- all organisms which allow expression of the nucleic acids according to the invention, their allele variants, their homologues, functional equivalents or derivatives, the recombinant nucleic acid construct or the vectors are suitable as host organisms.
- Host organisms are understood to mean, for example, eukaryotic or prokaryotic host organisms such as 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 humans or animals, for example HEK293, C0S1, C0S7, CHO, PC12, or SF9 cells.
- transgenic or recombinant organisms are characterized in that they either do not contain the nucleic acids according to the invention, their allele variants, their homologues, functional equivalents or derivatives, the recombinant nucleic acid construct or the vectors, or do not contain them at this point in the genome or in the cell or else that the natural promoter of the nucleic acid sequences according to the invention has been genetically manipulated in such a way that the corresponding genes are expressed more or less, depending on the situation.
- the prokaryotic or eukaryotic host organisms can contain at least one nucleic acid sequence according to the invention or at least one nucleic acid construct or at least one vector according to the invention.
- the gene product can also be used in transgenic organisms such as transgenic animals e.g. Mice, rats, sheep, cattle or pigs can be expressed. In principle, transgenic plants are also conceivable. The transgenic organisms can also be so-called knock-out animals.
- transgenic animals e.g. Mice, rats, sheep, cattle or pigs can be expressed.
- transgenic plants are also conceivable.
- the transgenic organisms can also be so-called knock-out animals.
- the transgenic animals can contain a functional or non-functional nucleic acid sequence according to the invention or a functional or non-functional nucleic acid construct.
- transgenic animals in their germ cells or in all or part of the somatic cells; or in its germ cells and all or part of the somatic cells the invention Nucleic acid sequence were changed by genetic engineering methods or interrupted by inserting DNA elements.
- the combination of the host organisms 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 ⁇ , Mu or other tempered phages or transposons and / or further advantageous regulatory sequences form a expression system.
- the expression systems are preferably understood to mean, for example, the combination of mammalian cells such as cells of neuronal origin and vectors such as pcDNA3 vectors or CMV vectors which are suitable for mammalian cells.
- the invention also relates to the use of the nucleic acids or parts thereof according to the invention for gene therapy in mammals, e.g. Man. Sequences complementary to the nucleic acids according to the invention or parts thereof can also be used for gene therapy. Gene therapy encompasses all forms of therapy which either introduce sequences according to claim 1 or 2 into the body or parts thereof, or influence the expression of sequences according to claims 1 and 2.
- Gene therapy encompasses all forms of therapy which either introduce sequences according to claim 1 or 2 into the body or parts thereof, or influence the expression of sequences according to claims 1 and 2.
- oligonucleotides e.g. Antisense or hybrid RNA-DNA oligonucleotides, with any modifications which contain parts of the sequences according to claims 1 and 2 can be used. Viral constructs containing a sequence according to claim 1 and 2 or parts thereof can also be used.
- Naked DNA containing a sequence according to claims 1 and 2 or parts thereof can also be used.
- Nucleic acid pieces with enzymatic activity e.g. ribozymes
- sequence polymorphisms that correlate with predispositions to human diseases can be detected in this way.
- Hereditary diseases which are characterized by hypoxic or ischemic cells or tissues can thus advantageously be detected.
- a further possibility of using the nucleotide sequence or parts thereof is the generation of transgenic or knock-out or conditional or region-specific knock-out animals or specific mutations in genetically modified animals [Ausubel et al. (eds), 1998, Current Protocols in Molecular Biology. John Wiley & Sons, (New York); Torres et al., 1997, Laboratory protocols for conditional gene targeting, Oxford University Press, (Oxford)].
- transgenic overexpression or genetic mutation zero mutation or specific deletions, insertions or changes
- animal models can be generated that provide full further information on the (patho-) physiology of the sequences according to the invention.
- a preferred embodiment consists in introducing the mutations of the A013 gene found in human hereditary diseases or polygenic inherited diseases into the germ line of transgenic animals. Animal models produced in this way can represent essential test systems for evaluating novel therapeutic agents that influence the function of A013.
- nucleic acid constructs or the corresponding proteins according to the invention proteins can be identified which have specific binding affinities for the protein according to the invention, or for the identification of nucleic acids which code for proteins which have specific binding affinities for the protein.
- 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 determined.
- an object of the invention is the use of the two-hybrid system or biochemical methods alone or in combination to identify the interaction domains of A013 and the use for pharmacotherapeutic intervention.
- Another particularly advantageous subject of the invention are protein complexes from the A013 protein and at least one further protein interacting with A013, such as Skpl.
- A013 gene as class I IEG is only expressed extremely low in the uninduced state.
- a very low basal A013 mRNA expression can be demonstrated in humans for the brain, placenta, kidney and heart (FIG. 3).
- the size of the A013 mRNA detected was approximately 4.0 kb in all organs.
- the experiments were normalized to S26, a ribosomal protein of the small subunit.
- MECS maximum electroconvulsive shock
- Ischemia (a valid animal model for human cerebral hypoxia, such as intranatal asphyxia, intraoperative hypoxia, cerebral hypoxia after cardiac arrest) shows that the sequences according to the invention play a role in global ischemia, the A013 mRNA expression can be induced in this animal model ( Figure 11, example 5).
- To induce global cerebral ischemia the internal carotid artery was clamped on both sides and, at the same time, hypotension was induced for a period of 3 to 5 minutes. This causes neuronal damage over a longer period of time, particularly in certain brain regions such as the hippocampus.
- Skpl and these so-called F-box proteins are part of a 5 multi-protein complex which is involved in the ubiquitin-dependent protein degradation (Hochstrasser, 1995, Curr. Opin. Cell Biol., 7: 215-223).
- This complex is called SCF complex (Skpl-Cullinl-F-Box protein) and is a component of the ubiquitin-ligase complex (E3) [Bai et al., (1996), Cell, 10 86: 263-274; Skowyra et al., (1997) Cell 91: 209-219; Deshaies R.J., (1999), Annu. Rev. Cell Dev. Biol. 15: 435-67].
- A013 appears to be another representative of the F-Box proteins.
- ubiquitin molecules 15 are transferred to a protein substrate in a multi-stage reaction (E1-E3) (FIG. 8).
- E1-E3 multi-stage reaction
- the degradation of ubiquitinated protein substrates then takes place on 26S proteasomes, which can be located both in the cell nucleus and in the cytoplasm [Ciechanover A. (1994), Cell, 79: 13-21; Jentsch S., (1992), Annu. Rev. Genet., 20 26: 179-207].
- the degradation process described above must be both highly selective and strictly regulated so that the large number of individual proteins can be degraded as required in accordance with their half-lives, which can range from a few minutes to several days.
- Ubiquitin-dependent protein degradation is used to determine the cellular concentration of a large number of important regulatory
- proteins controlled These proteins play a key role in processes such as cell cycle control, cell differentiation, DNA repair mechanisms and the control of various signal transduction cascades.
- denatured and aggregated proteins are also above this
- Ubiquitin a protein consisting of 76 amino acids, is first activated by an ubiquitin-activating enzyme (El) in an ATP-dependent reaction.
- the activated ubiquitin is transferred as a thioester to an ubiquitin-conjugating enzyme (E2).
- E2 ubiquitin-conjugating enzyme
- E3 ubiquitin ligase activity
- Multiple ubiquitin residues can also be transferred to the protein substrate at the E3 complex, which leads to polyubiquitination of the target protein.
- the ubiquitinated protein substrates are recognized and broken down by the 26S Proteasome.
- the F-Box proteins play a key role in the ubiquitination of target proteins.
- A013 is the only known IEG that, as an F-Box protein, controls the degradation of specific target proteins through ubiquitin-dependent protein degradation.
- the proteins that are recruited to interact with A013 to form the E3-ubiquitin-ligase complex are still unknown. Due to the nuclear localization of the A013, transcription factors or other nuclear regulatory proteins would be conceivable as interaction partners, which should be removed from the cell metabolism after cellular stress, as induced by convulsive states or ischemic or hypoxic states.
- F-box proteins are generally of high medical relevance.
- the absence of an F-Box protein or a non-functional F-Box protein can occur due to the resulting accumulation of the associated protein substrates, associated with pathological changes.
- a number of clinical pictures are known which are caused by mutations in the ubiquitin-conjugating system (or which are associated with a disturbed protein degradation on the 26S proteasome).
- UBE3A ubiquitin protein ligase 3A gene
- E6AP ubiquitin protein ligase 3A gene
- UBE3A is a 100 kDa protein that interacts with the E6 protein of the human papillomavirus type 16 and 18.
- the UBE3A-E6 complex recruits the tumor suppressor p53 to form the E3 ubiquitin ligase complex.
- Angelmann syndrome is characterized by mental retardation,
- Liddle syndrome is a hereditary form of arterial hypertension, which is characterized by very low serum aldosterone, renin and angiotensin levels. (Liddle et al., (1963),
- Nedd4 is an ubiquitin ligase that controls the degradation of the epithelial Na + ion channel (Anan et al., 1998; Genes Cells, 3: 751-763; Staub et al., 1996, EMBO J. 15: 2371-2380). Interaction with 0 the so-called proline tyrosine motif (PYXY) of the epithelial
- Na + ion channel takes place via the WW domains of Nedd4 (Staub et al., 1996, EMBO J. 15: 2371-2380.
- pVHL von Hippel-Lindau protein
- HIF-1 hypoxia-inducible transcription factors
- PVHL thus fulfills a function analogous to that of F-Box proteins.
- the constitutive expression of the genes regulated by HIFs as well as the expression of the vascular endothelial growth factor (VEGF) determines the hypervascularization and proliferation of the surrounding tissue (Kaelin and Gurher, 1998, Trends Genet. 14: 423-426; Pause et al., 1998, Proc. Natl. Acad. Sci. 95: 993-998).
- VEGF vascular endothelial growth factor
- Treat diseases that are associated with impaired protein breakdown include seizure disorders (epilepsy) and neurodegenerative diseases such as cerebral ischemia, Parkinson's disease, Huntington's disease, Alzheimer's disease or CNS trauma.
- seizure disorders epilepsy
- neurodegenerative diseases such as cerebral ischemia, Parkinson's disease, Huntington's disease, Alzheimer's disease or CNS trauma.
- Mutations that affect the functionality of the A013 can be of various types. These can either be mutations of larger areas or smaller changes in the nucleic acid sequence. Examples of both options are familiar to the person skilled in the art and include deletions, insertions, inversions, rearrangements which relate to the nucleic acid sequence of A013, but also base exchanges / point mutations.
- the mutations can change the region of A013 coding for the protein sequence. This can lead to changes in the protein sequence (substitution, inversion, insertion or deletion of one or more amino acid residues) and thus to new properties of the modified protein (gain-of-function mutation).
- mutations can affect the 5 'or 3' untranslated region of the A013 gene, change (flanking) regulatory sequences (e.g. promoters, intronic sequences, splice sequences, enhancers, silencers, locus control regions, matrix attachment regions, etc.) or delete.
- Dysregulation of expression can also lead to hypomorphic alleles of A013. Complete or partial deletion, insertion or rearrange ent from the A013 locus can occur, which would lead to a loss of function of the gene (loss-of-function). Mutations that result in the use of open reading frames not used in the wild-type allele are also possible. Chromosomal mutations can also lead to the recombination of new functional units.
- a fusion transcript can be created from parts of A013, recombined with a second gene.
- This fusion gene can code for a protein with new properties.
- the coding sequence of the one fusion partner eg without being changed in the sequence itself under the control of regulatory elements of the second partner.
- SSCP single stranded conformation polymorphism
- Structural analyzes of the protein (s) according to the invention specifically allow substances to be found which have a specific binding affinity.
- sequences SEQ ID NO: 1, SEQ ID NO: 3 and / or SEQ ID NO: 5 described enable, with the aid of 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.
- Amino acid sequence or a protein complex containing the protein with the amino acid sequence according to the invention by expression of the nucleic acid according to the invention, the expression cassette or the vector in a eukaryotic or prokaryotic organism. b) incubation of the protein or proteins with the substance to be tested.
- Binding is detected by measuring the antagonization or agonization of A013 activity or by measuring the physiological effect of A013.
- Further embodiments of the invention are a method for finding substances which inhibit or intensify the interaction of ligands with the protein heteromer or the proteins according to the invention or a method for finding substances which interact with the proteins of the invention with proteins such as Skpl or other interaction partners inhibit or reinforce.
- the interaction of proteins with the amino acids according to the invention can be detected, for example, using the two-hybrid system or by biochemical methods such as, for example, by co-immunoprecipitation.
- the methods can be carried out by expression of the proteins in eukaryotic cells and linkage with a reporter assay for the activation of the A013 protein.
- the invention relates to a method for the qualitative and quantitative determination of proteins with the amino acid sequence according to the invention using specific agonists or antagonists.
- the A013 ligand binding is used for detection.
- the protein activity or amount of the proteins according to the invention can be determined via antibodies.
- the invention therefore furthermore relates to a method for quantifying the protein activity or amount of the protein according to the invention with the sequences SEQ ID NO: 2, SEQ ID NO: 4 and / or SEQ ID NO: 6 or its functional equivalents or homologues.
- 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 A013 and others Genes are used. This results in the possibility of gene expression of nucleic acid constructs using the A013 promoter in the dentate gyrus.
- the region before the start of transcription is first of all subjected to a reporter gene such as e.g. ⁇ -galactosidase or GFP (Green Fluorescent Protein) linked and in cells or in transgenic animals such as e.g. Mice then tested whether it leads to the expression pattern specific for sequence SEQ ID NO: 1 and SEQ ID NO: 3 or SEQ ID NO: 5 (Ausubel et al., (Eds), 1998, Current Protocols in Molecular Biology. John Wiley & Sons, New York).
- a reporter gene such as e.g. ⁇ -galactosidase or GFP (Green Fluorescent Protein) linked and in cells or in transgenic animals such as e.g. Mice then tested whether it leads to the expression pattern specific for sequence SEQ ID NO: 1 and SEQ ID NO: 3 or SEQ ID NO: 5 (Ausubel et al., (Eds), 1998, Current Protocols in Molecular Biology. John Wiley & Sons, New York).
- cis-regulatory sequences inter alia can be located at a very large distance 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, e.g. BACs or YACs (Bacterial artificial chromosome, yeast artificial chromosome) to use.
- the reporter gene can be inserted into the vector via homologous recombination and its expression can be examined (see, for example, Hiemisch et al., 1997, EMBO J. 16: 3995-4006).
- suitable deletions in the construct and subsequent investigation of the effects on the expression of the reporter gene important regulatory elements can be identified (see e.g.
- the regulatory sequences of the nucleic acids according to the invention can be used for the tissue-specific expression of the nucleic acids according to the invention and other genes. This results in the possibility of predominantly gene expression in the dentate gyrus of nucleic acid 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, J. 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.
- synthetic peptides can be generated which are used as antigens for the production of antibodies. It is also possible to use the polypeptide or fragments thereof to generate antibodies.
- Antibodies are both 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 are in principle to be understood as meaning all immunoglobulin classes such as IgM, IgG, IgD, IgE, IgA or their subclasses such as the subclasses of the IgG or their mixtures.
- IgG and its subclasses such as IgG ⁇ , IgG, IgG 2a I ⁇ ? G b , IgG 3 or IgGii are preferred.
- the IgG subtypes IgG ⁇ / ⁇ 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 chain 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-strand fragments such as Fv, Fab or F (ab ') are preferred.
- 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 manipulated, unabridged fragments can also be used advantageously.
- 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. (eds), 1998, Current Protocols in Molecular Biology. John Wiley & Sons, (New York); Harlow and Lane (eds), 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, (New York)].
- antibody-producing cells are attracted and the mRNA is isolated from the cells in a known manner if the optical density of the cells is sufficient via cell lysis with guanidinium thiocyanate, acidification with sodium acetate, extraction with phenol, chloroform / isoamyl alcohol, precipitation with isopropanol and washing with ethanol.
- the reverse transcriptase is used to synthesize cDNA from the mRNA.
- the synthesized cDNA can be inserted directly or after genetic manipulation, for example by "site directed mutagenesis", introduction of insertions, inversions, deletions or base exchanges into suitable animal, fungal, bacterial or viral vectors and expressed in the corresponding host organisms.
- Bacterial or yeast vectors such as pBR322, pUCl8 / 19, pACYC184, lambda or yeast mu vectors are preferred for cloning the genes and for expression in bacteria such as E. coli or in yeast such as Saccharomyces cerevisiae.
- Specific antibodies against the proteins according to the invention can be suitable both as diagnostic reagents and as therapeutic agents for clinical pictures which are characterized, inter alia, by changes in neurons of the dentate gyrus.
- 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 develop a test system.
- 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 the proteins according to the invention or on a complex consisting of at least one of the proteins according to the invention and a further protein (e.g. Skpl).
- the determination of the amount, activity and distribution of the proteins according to the invention, its functional equivalents or homologues or the underlying mRNA in the human body can serve for diagnosis, detection of the predisposition and for monitoring in certain diseases.
- the nucleic acid sequences according to the invention and the 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 serves according to Claim 1 or parts thereof in the form of suitable samples for detecting point mutations or deletions / insertions / rearrangements.
- the proteins shown in Example 1, 2 or 2 and reagents derived therefrom (oligonucleotides, antibodies, peptides) can be used for the diagnosis and therapy of 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.
- Another object of the invention is 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 u:
- 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 which comprises one or more of the steps mentioned below for finding substances which bind specifically to a protein having the amino acid sequence according to the invention or to a protein complex which contains at least one protein according to the invention and at least one further protein which contains the protein of the invention interacts:
- Amino acid sequence or a protein complex according to the invention by expression of a nucleic acid according to the invention, an expression cassette or a vector in a eukaryotic or prokaryotic organism,
- 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 also relates to a method for finding substances which bind specifically to the protein or protein complex according to the invention and thereby cause inhibitory or activating functional effects on the function of the A013, preferably in neurons.
- Nucleic acid ie 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 A013 gene.
- suitable substances can be found, for example, by determining their effect on the transcription elements of the A013 gene.
- specific, synthetic or natural, competitive and non-competitive antagonists against the protein, or antibodies or antibody fragments against the protein or against the protein heteromer can be used.
- inhibition of the A013 activity or the activity of the protein can be achieved both by antisense molecules or ribozymes or oligonucleotides and by low molecular weight compounds.
- Short nucleic acid fragments of 15 to 100 nucleotides, preferably 15 to 40 nucleotides, particularly preferably 15 to 25 nucleotides, are advantageously used as antisense molecules.
- nucleic acids according to the invention or complementary nucleic acid sequences derived therefrom can advantageously be used for the production of medicaments for the treatment of diseases of ischemia, which can be influenced positively by modulating the gene expression of A013.
- 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 A013 protein.
- Substances that 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 positively influenced by modulating the activity or amount of A013 protein.
- the diseases mentioned can be neurodegenerative diseases such as epilepsy, ischemia, dementia, Parkinson disease, Huntington disease, Alzheimer's disease or CNS trauma. Diseases such as epilepsy, cerebral ischemia, M. Alzheimer's or CNS trauma are preferred.
- nucleic acid sequences of 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 for example, so-called tags are known in the literature (for example the hexa-histidine anchor) or epitopes which can be recognized as antigens of various antibodies (described for example in Harlow & Lane (eds), 1988, Antibodies: Laboratory Manual, Cold Spring Harbor Press, New York).
- These 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.
- a solid support such as a polymeric matrix
- 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 to derivatize the proteins.
- the nucleic acids according to the invention can also be used as markers for human or animal inherited diseases or for gene therapy.
- 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 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.
- the following examples describe the rat A013 cDNA sequence (SEQ ID NO: 1), the human form (SEQ ID NO: 3), and parts of the mouse genomic sequence (SEQ ID NO: 5).
- the corresponding A013 amino acid sequences of the rat, human and mouse are listed under SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6, respectively.
- Figure 1 Development-specific expression in the rat (in situ hybridization)
- Cere Cerebellum; Ent: entorhinal cortex; Hi: hippocampus; Olf: Bulbus Olfactorius; Pir: Cortex Piriformis; Tha: thalamus)
- FIG. 1 Northern blot analysis: tissue-specific expression in the rat
- Subunit normalized (mm: mus musculus).
- FIG. 4 In situ hybridization: brain sections (after cain administration)
- A013-myc-His was detected with an anti-myc antibody and a Cy3-conjugated secondary antibody.
- Figure 6 Schematic representation of the A013 domain structure
- Figure 7 Homology comparison: A013 F-Box domain with F-Box Motif (from PFAM database)
- a Pfam database search revealed homology from A013 to F-box proteins. Strongly preserved amino acids of this motif are highlighted in gray. In A013 and in the F-Box motif matching amino acids are identified by (:) and similar amino acids by (.).
- E3 ligase complex The components of the E3 ligase complex are highlighted in light gray (El: ubiquitin activating enzyme; E2: ubiquitin conjugating enzyme; E3: ubiquitin ligase (shown in dark gray); here consisting of: SCF-
- Skpl- Cullinl - F Box Protein Skpl: S-phase kinase associated protein 1; Culll: Cullin 1; Practice: ubiquitin
- Polyclonal antibodies were produced against the region of amino acid 95-184 of rA013 and tested by Western blot analysis.
- Cell extracts from A013-myc-His transfected cells and the corresponding vector control were used.
- the signals for a commercially available anti-myc antibody, the preimmune serum and the A013 immune serum obtained are shown.
- the A013 immune serum shows a specific band of the expected size of approximately 60 kDa.
- a hippocampus ⁇ phage library (primed by rats after MECS stimulation and administration of cycloheximide; oligo (dT), in UniZAP, Stratagene) was screened with the above-mentioned A013 cDNA fragment as a probe under standard conditions.
- the clones positive in the hybridization were separated, subcloned and subjected to a sequence analysis.
- the nucleic acid sequence SEQ ID NO: 1 was obtained for the A013 of the rat.
- the ORF codes for a protein of 563 amino acids (SEQ ID NO: 2).
- BLS [BLASTN 2.0.11, Atschul et al., (1997), Nucleic Acid Res, 25, 3389-3402] were used to determine related ESTs in rats and humans.
- the 3 'primer 5' -ACAATAGAGCAGCCAGGTCTC-3 ' was derived from the human EST AI608739.
- the sequence segment 5 '-CTGCATGCACCTGTTTCACTG-3' from the coding region of the rat cDNA was used as a primer as the 5 'primer.
- a 2.4 kb PCR fragment was amplified with the aforementioned pimers by means of PCR using human hippocampus cDNA as a template.
- a human hippocampus cDNA library (oligodT and randomly primed, in Lambda ZAP, Stratagene) was searched under standard conditions. The clones positive in the hybridization were separated, subcloned and subjected to a sequence analysis. Here was obtained the nucleic acid sequence SEQ ID NO: 3 for human A013.
- the ORF codes for a protein of 555 amino acids (SEQ ID NO: 4).
- A013 cDNA clone (A013 / 17; contains the complete cDNA sequence of the rat), a 2.5 kb fragment was isolated using the restriction enzyme Xhol, which contains the main part of the coding A013 region. This fragment was radioactively labeled and used to hybridize a mouse genomic cosmid library (129 / Ola, RZPD, Berlin). With the help of a Southern blot, six positive clones were found (A3, B5, B6, C3, C6, Dl).
- a 10 kb fragment from the A013 cosmid C3 (MPMGcl2lBl3681Q2) was subcloned into a plasmid vector and sequenced by means of a transposon insertion method (GPS-1, New England Biolabs, Beverly, MA; USA), and the sequences with the program SeqMan ( Lasergene, Madison, WI, USA) compared.
- GPS-1 New England Biolabs, Beverly, MA; USA
- SeqMan Lasergene, Madison, WI, USA
- a construct was used as template for in situ hybridizations, which contained the rA013 cDNA via EcoRI and Xhol linker in the vector pBlueScript (pBS).
- pBS vector pBlueScript
- the plasmid was linearized with AccI, which resulted in a 1585 bp A013 cRNA fragment during transcription.
- the plasmid was linearized with Styl, which resulted in a 1107 bp long rA013 cRNA fragment. From these templates, A013-specific sense and antisense cRNA samples were generated using T3 RNA polymerase and T7 RNA polymerase, digoxigenin-labeled UTP and standard NTPs manufactured.
- Frozen rat brains and embryos were made at -20 ° C in 15 ⁇ m sections on the cryostat (Leica GmbH), fixed with 4% paraformaldehyde in PBS, permeabilized in 0.2% Triton-X 100 and with antisense and sense A013 cRNA samples hybridized at 65 ° C overnight in 50% formamide, 5X SSC. The sections were washed with 0.2X SSC at different temperatures and the dioxigenin label was detected with a specific antibody (Kuner et al., 1999, Science, 283: 74-77).
- In situ hybridizations (embryo day 19; Figure 1) and Northern blot analyzes of rat brains of different ages (embryo day 9.5 to adult; not shown) showed that rA013 is hardly expressed both embryonic and in the adult stage (not shown). Whereas A013 is detectable during postnatal development (in situ hybridizations from day 4 and day 7) in the hippocampus, in the cerebellum, in the temporal, parital and entorhinal cortex, in the amygdala, in the striatum, in the olfactory bulb and in the brain stem.
- Northern blot analyzes of various tissues from adult rats showed no significant expression of the A013 mRNA.
- the A013 mRNA was only detectable to a very small extent for the brain, lungs, kidneys and intestine.
- the size of the A013 mRNA detected was approximately 4.0 kb in all organs (FIG. 2).
- the A013 mRNA expression pattern in human tissues was analyzed.
- a blot with poly (A) + RNA from 12 different organs (Clontech) was hybridized with radioactive probes for rA013 and S26, a ribosomal protein of the small subunit, according to standard methods.
- radioactive probes for rA013 and S26 a ribosomal protein of the small subunit, according to standard methods.
- only signals of very weak signal strength were received for the brain, placenta, kidney and heart.
- the size of the human A013 mRNA detected was also approximately 4.0 kb (FIG. 3).
- Example 5 Expression analysis of A013 after application of kainate, PTZ and after global ischemia
- the expression of the A013 mRNA is strongly induced after antispasmodic stimuli such as multiple MECS with administration of cycloheximide and after administration of convulsive doses of kainate [Cole et al., 1990, J. Neurochem. 55: 1920-1927; Worley et al., 1993, J. Neurosci 13: 4776-4786; Lanahan and Worley, 1998, Neurobiol. Learn. Mem. 70: 37-43; WO99 / 40225: Worley et al., 1999]. In this experiment it was examined whether the A013 mRNA expression also in other neurodegenerative processes, such as after global ischemia.
- 3-NPA induces mild cellular hypoxia by inhibiting succinate dehydrogenase. 3-NPA may therefore induce the transcription of the A013 gene via an indirect generation of reactive oxygen species.
- A013 mRNA expression was predominantly restricted to this region of the brain, which plays an important role in learning processes, memory, synaptic transmission and neuronal plasticity.
- rA013 was provided with a carboxy-terminal c-Myc tag, expressed heterologously in mammalian cells and then the location of the overexpressed protein was determined by immunofluorescence analysis 0
- the coding region of the rA013 cDNA was cloned into the vector pcDNA3.1 / Myc-His A (Invitrogen), which allows the desired gene product carboxy-terminal to be provided with a c-myc-6xHis tag.
- pcDNA3.1 / Myc-His A Invitrogen
- a PCR fragment generated with the primers rA013-5 '-Notl-ATG 5 and rA013-3'-HindIII-558, which codes for amino acids 1-558, and an oligonucleotide linker which was used for the remaining 5 Amino acids encoded and both cloned in the above vector.
- PCR primer rA013-5 '-Notl-ATG: 5' -AGCTTCAGCGAAGACCATTT-3 '-.
- rA013-3'-HindIII-558 5 '-CTAGAAATGTCTTCGCTGA-3'
- COS-1 cells were cultured in high glucose-DMEM medium (Sigma Immunochemicals), 10% fetal calf serum (Sigma Immunochemicals) at 37 ° C, 8% CO 2 and 95% atmospheric humidity and according to DEAE / Dextran DNA transfection method transfected with pcDNA3.l-Myc / His-rA013. 48 hours after transfection of the cells with rA013-myc or the pcDNA3.1 / Myc-His A vector, the cells were washed with PBS and fixed twice with 3% paraformaldehyde in PBS (Sigma Chemicals) for 15 min 5. The fixation was stopped by two 15-minute incubations with 50 M NH 4 CI-PBS.
- the cells were permeabilized with 0.2% Triton-X-100 in PBS (Sigma Chemicals) and then blocked with 1% BSA in TBS (20 mM Tris-HCl; 20 mM NaCl; 0.5 mM KC1; 1 mM MgCl 2 ; 0.02% NaN 3 ; pH 9.0) for 30 min. After incubation for 1.5 hours with a monoclonal anti-c-Myc antibody (Invitrogen) in 1% blocking solution at room temperature, the cells were washed three times for 5 min each with TBS and for one hour with Cy3-labeled anti-mouse antibody (Jackson Laboratories Inc .; 15 ⁇ g / ml in blocking solution).
- the cells were washed three times for 10 min each with TBS and three times with 10 mM Tris-HCl, pH 7.6.
- the coverslips were then sealed with aqueous mounting medium (Sigma Chemicals).
- the preparations were analyzed under the fluorescence microscope (Zeiss Axiophot) with a standard filter set.
- the rA013-myc had a nuclear localization ( Figure 5).
- the empty control plasmid showed no specific signals ( Figure 5). This observation is in agreement with the presence of a nuclear localization signal "(PDGKKIK) in amino acid position 238 to 244 (FIG. 6) of rA013.
- rA013 has a strongly basic, arginine-rich domain at the amino terminal (1-61 AS; Arg content 35%; 51% basic AS), followed by a putative F-box domain (AS 73-114) (FIG. 6).
- a putative leucine zipper extends from amino acid 177 to 204 and is followed by the hydrophobic leucine-rich C-terminus of the protein (FIG. 6).
- a potential nuclear localization signal (amino acids 238-244) is detectable, which could be responsible for the transport of A013 in the cells.
- the two-hybrid screening was carried out with the ProQuest TM Two Hybrid System from Gibco BRL.
- the cDNA fragment coding for the F-box domain of the A013 gene (SEQ ID NO 1; amino acids 70-114, SEQ ID NO: 2) with the specific A013 primers A013-5 '-Y2H-70 (5' - AGATGTCGACCGGCGCCGCGTCGCTGCCC-3 ') and A013-3' Y2H-114 (5 '-ACTTTAGCGGCCGCTTAGAGCTGGGGCCACAGGGC-3') were amplified in a PCR (polymerase chain reaction), and after restriction digestion with the enzymes Notl and B, the Gibco p1 and the enzymes Notl and Sb ) cloned.
- the resulting construct pDBLeu-rAO13 (70-114 AS) codes for a fusion protein from the GAL4 binding domain that is fused at the C-terminal to the F-box domain of rA
- the construct pDBLeu-rAO13 (70-114 AS) obtained was transformed into the yeast strain Y190.
- the concentration of 3-aminotriazole (3AT) required for the inhibition of the basal expression of the HIS3 gene was determined for the transformed yeast strain.
- HIS3 codes for the imidazole glycerol phosphate dehydratase, an enzyme of histidine biosynthesis. In the presence of 3AT in the determined limit concentration, the growth of yeast cells that have no two-hybrid interactions is suppressed.
- the pDBLeu-rA013 (70-114 AS) yeast strain was transformed with a rat hippocampus / cortex cDNA library from rats stimulated with MECS (see WO99 / 40225: Worley et al., 1999).
- Vector system for the cDNA bank the vector pPC86 (Gibco BRL) was used, this enables expression as a fusion protein with the GAL4 DNA activation domain.
- the pPC86 plasmid DNA was purified from the positive yeast colonies and transformed into the E. Coli strain XLl-Blue for amplification.
- the pPC86 plasmid DNA of the positive colonies obtained was co-transformed into the yeast strain Y190 using various pDBleu constructs.
- Skpl S-phase-kinase-associated protein
- a 6x-His-A013 fusion protein was used to produce polyclonal antibodies against the A013 protein of the rat.
- a PCR fragment using the primers 5 '-N-BamHI (5' -GCGGATCCTCGGCCTCCTGCTCGCACTGG-3 ') and 3' -N-Hind III (5'-CCCAAGCTTGGAGGAGTTGCAGGGC-3 ') generated for the amino acids 95 to 184 encoded and cloned into the expression vector pQE30 (Qiagen).
- Expression of the AO13 fusion protein in E. coli was induced with 200 UM IPTG for 5 hours at 37 ° C.
- the cells were then lysed and the 6x-His-rA013 (95-184 AS) extracted with a 6 M urea solution and purified to homogeneity by means of nickel-chelate affinity chromatography.
- the fusion protein was injected into rabbits for antibody production (carried out by Eurogentec).
- the immune serum obtained was tested by Western blot analysis for a specific reaction with heterologously expressed rA013 protein.
- HEK293 cells were transiently transfected with an rA013 expression construct consisting of the open reading frame of rA013 fused to a C-terminal c-myc / His tag.
- the cells were harvested after 48 hours, lysed and the protein extract was separated into triplicates on a denaturing protein gel and blotted.
- a Western blot analysis with the immune serum showed - compared to the preimmune serum - a specific signal of the expected size of approximately 60 kDa. ( Figure 9).
- an anti-myc antibody Invitrogen, a band of the same size was obtained.
- the mouse genomic sequences obtained from A013 are shown in FIG.
- the targeted mutation of the A013 gene in the mouse germ line and the analysis of the resulting phenotype can provide important additional information about the (patho) physiological mechanisms in which the A013 gene is involved.
- a so-called knock-out mouse ie a mouse without a functional A013 protein
- a so-called targeting construct was first produced.
- Two genomic fragments flanking the coding region of A013 (corresponding to positions 1 bp to 1148 bp and 1760 bp to 7425 bp) in the sequence SEQ ID NO: 3 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) cloned.
- 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 A013 and was thus under the control of the endogenous A013 promoter.
- the lacZ cassette replaces exon 1 of A013 ( Figure 10).
- the DNA can be electroporated into embryonic stem cells in a manner known to those skilled in the art and selected for G418-resistant clones.
- Genomic DNA can be isolated from these clones and tested for homologous recombination between targeting construct and endogenous A013 allele using so-called nested PCR.
- FIG. 10 shows the genomic structure of A013, the homology arms chosen for homologous recombination, and possible primers for the PCR detection of homologous recombination. Only a PCR product can be created after homologous recombination of the A013 targeting construct.
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WO1999018989A1 (en) * | 1997-10-16 | 1999-04-22 | Baylor College Of Medicine | F-box proteins and genes |
WO1999040225A1 (en) * | 1998-02-09 | 1999-08-12 | The Johns Hopkins University School Of Medicine | Immediate early genes and methods of use therefor |
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WO1999018989A1 (en) * | 1997-10-16 | 1999-04-22 | Baylor College Of Medicine | F-box proteins and genes |
WO1999040225A1 (en) * | 1998-02-09 | 1999-08-12 | The Johns Hopkins University School Of Medicine | Immediate early genes and methods of use therefor |
Non-Patent Citations (1)
Title |
---|
SKOWYRA D ET AL: "F-Box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex" CELL, CELL PRESS, CAMBRIDGE, NA, US, Bd. 91, 17. Oktober 1997 (1997-10-17), Seiten 209-219, XP002189329 ISSN: 0092-8674 * |
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