WO2001057208A2 - Mlp-gen, nukleinsäuren, polypeptide und deren verwendung - Google Patents
Mlp-gen, nukleinsäuren, polypeptide und deren verwendung Download PDFInfo
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- WO2001057208A2 WO2001057208A2 PCT/EP2001/001042 EP0101042W WO0157208A2 WO 2001057208 A2 WO2001057208 A2 WO 2001057208A2 EP 0101042 W EP0101042 W EP 0101042W WO 0157208 A2 WO0157208 A2 WO 0157208A2
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
Definitions
- MLP gene nucleic acids, polypeptides and their use
- the present invention relates to nucleic acids coding for MLP, a polypeptide encoded thereby, probes for these nucleic acids, methods for the detection of and / or screening for myocardial diseases, kits for the detection of the nucleic acids and the polypeptide, regulatory nucleic acids, these comprehensive vectors, these comprehensive Cells and this comprehensive medication.
- DCM Dilated cardiomyopathy
- HCM hypertrophic cardiomyopathy
- the MLP knock out which is associated with a DCM, fits well into the thought model mentioned above (Arber et al .; Cell 88, 393-403 (1997)).
- the MLP was first described in 1994 using a subtractive cloning technique as a positive regulator of myogenesis (Arber S. et al .; Cell 79, 221-31 (1994)) and gained considerable importance in cardiology in 1997 when it became known (Arber et al .; Cell 88, 393-403 (1997)) that the MLP knock out in a mouse model is accompanied by a DCM. This was also the first genetically engineered organism to develop this clinical picture. However, it is still completely unclear in the prior art whether mutations in this gene also lead to DCM in humans.
- the MLP itself belongs to the LIM protein group. This group was named about 10 years ago after the initial letters of its first 3 members (lin 11, islet 1, mec3) and has essentially three subgroups: 1.
- the MLP belongs to the group of “LIM only” proteins (Morgan, M. et al .; Biochem Biophys Res Com 212, 840-846 (1995)).
- the protein itself consists of 194 amino acids and forms two LIM double zinc fingers, each followed by a glycine-rich amino acid sequence.
- the protein probably acts due to its location on the Z band as a cytoskeletal protein, where it binds to f-actin structures (Arber, S. et al .; Genes Dev 10, 289-300 (1996)).
- the present invention has for its object to provide an agent that allows preventive treatment of dilated cardiomyopathy.
- the remedy is also intended to demonstrate the disposition of an individual to develop the clinical picture of the dilated Allow cardiomyopathy.
- the agent to be provided should enable the diagnosis of dilated cardiomyopathy.
- the invention is based on the object of providing a regulatory nucleic acid which allows the tissue-specific, in particular muscle-specific, expression of nucleic acids.
- nucleic acid which codes for an MLP and comprises a sequence according to SEQ ID NO: 1, the sequence having a mutation at base no. 10 of exon 2 of the translated sequence.
- the mutation is an exchange from T to C.
- nucleic acid which codes for an MLP and comprises a sequence according to SEQ ID NO: 1, the sequence having a mutation at the third position of codon 112 in exon 4 of the translated sequence.
- the mutation is an exchange from A to G.
- the nucleic acid only comprises the sequences coding for an amino acid sequence.
- nucleic acid which codes for an MLP and which comprises the sequence from positions 58 to 639 of SEQ ID NO: 1, a mutation being present at position 67.
- the mutation is a mutation from T to C.
- nucleic acid which codes for an MLP and comprises the sequence from positions 58 to 639 of SEQ ID NO: 1, a mutation being present at position 393.
- the mutation is a mutation from A to G.
- nucleic acid coding for MLP the sequence would correspond to a nucleic acid according to the invention without the degeneracy of the genetic code.
- nucleic acid coding for MLP the nucleic acid hybridizing with a nucleic acid according to the invention.
- the object is achieved according to the invention by an amino acid sequence which is encoded by a nucleic acid according to the invention or parts thereof.
- the amino acid sequence according to the invention is encoded by the sequence (s) of the nucleic acid according to the invention or parts thereof, which one or both of the aforementioned mutations, i.e. at Base No. 10 of exon 2 of the translated sequence or at the third position of codon 112 in exon 4 of the translated sequence.
- the object is achieved according to the invention by an MLP comprising an amino acid sequence which is encoded by a nucleic acid according to the invention or a part thereof, or by the MLP comprising an amino acid sequence according to the invention.
- the object is achieved according to the invention by a probe for a nucleic acid according to the invention, the probe comprising a sequence according to SEQ ID NO: 4.
- the probe is used for the detection and / or amplification of the nucleic acid according to the invention.
- the object is achieved according to the invention by a probe for a nucleic acid according to the invention, the probe comprising the sequence according to SEQ ID NO: 5.
- the probe is used for the detection and / or the amplification of the nucleic acid according to the invention.
- nucleic acids and / or the probes according to the invention are used to diagnose and / or screen for myocardial diseases.
- myocardial disease is dilated cardiomyopathy.
- the object is achieved according to the invention by a method for the detection of and / or screening for myocardial diseases, a nucleic acid according to the invention and / or a probe according to the invention being used.
- the myocardial disease is dilated cardiomyopathy.
- the object is achieved according to the invention by a method for the detection of and / or screening for myocardial diseases, wherein a sample comprising a sequence coding for MLP is digested with a restriction enzyme and the presence of a mutated coding coding for MLP by the occurrence a restriction enzyme digestion pattern changed compared to the restriction enzyme digestion pattern of a non-mutated sequence coding for MLP is detected.
- the myocardial disease is dilated cardiomyopathy.
- the method is an embodiment of the method according to the invention for the detection of and / or screening for myocardial diseases, wherein one nucleic acid according to the invention and / or a probe according to the invention is used.
- the sequence coding for MLP is amplified by means of PCR before digestion.
- the detection or screening is directed to a nucleic acid according to the invention.
- the amplification is carried out with a primer, the primer comprising a sequence according to SEQ ID NO: 13 and / or SEQ ID NO: 14.
- restriction enzyme digestion is carried out by Nci I.
- the PCR product in the case of a non-mutated sequence has a length of approximately 308 bp and in the case of a mutation at base no. 10 of exon 2 or a mutation at position 67 of the nucleic acid sequence of SEQ ID NO: 1, where the mutation is an exchange from T to C, two PCR products with approximately 205 and approximately 103 bp occur.
- the detection or screening is directed to a nucleic acid according to the invention, preferably to a nucleic acid which has a mutation at the third position of codon no. 112 in exon 4 of the translated sequence from SEQ ID NO: 1 has.
- the amplification is carried out with a primer, the primer comprising the sequence according to SEQ ID NO: 15 and / or SEQ ID NO: 16.
- the restriction enzyme is digested by Cvi Rl.
- a probe according to the invention for the detection of and / or screening for myocardial diseases, in particular dilated cardiomyopathy, in which a probe according to the invention is used, it can be provided that for the detection of or the screening for a nucleic acid sequence according to the invention, a hybridization of one to be examined Sample is carried out with a probe, the probe comprising a sequence according to SEQ ID NO: 4.
- the hybridization takes place at 60 ° C.
- a probe according to the invention for the detection of or the screening for a nucleic acid sequence according to the invention a hybridization of a sample to be examined done with a probe, the probe comprising a sequence according to SEQ ID NO: 5.
- the object of the invention is achieved by a method for the detection of and / or screening for myocardial diseases, an amino acid sequence according to the invention or an MLP according to the invention being detected.
- the myocardial disease is dilated cardiomyopathy.
- the amino acid sequence or the MLP is detected by means of an antibody.
- the antibody is a monoclonal antibody.
- the object is achieved by a kit for detecting a nucleic acid according to the invention, it being provided that the kit comprises at least one nucleic acid according to the invention and / or at least one probe according to the invention.
- the object is achieved according to the invention by a kit for detecting an MLP according to the invention or for detecting an amino acid sequence according to the invention coding for an MLP, the kit comprising at least one antibody against the MLP according to the invention or against an amino acid sequence according to the invention.
- the object is achieved by a regulatory nucleic acid sequence which comprises approximately 500 base pairs, the 500 base pairs being those which are arranged upstream of the first base of the first exon of the human genomic sequence coding for MLP.
- the regulatory nucleic acid comprises approximately 1000 base pairs, the 1000 base pairs being those which are arranged upstream of the first base of the first exon of the human genomic sequence coding for MLP.
- the object is achieved by a regulatory nucleic acid which can be represented on the basis of human genomic DANN using two primers in a PCR reaction, the upstream primer comprising SEQ ID NO: 9 and the downstream primer SEQ ID NO: 10.
- the object is achieved by a regulatory nucleic acid sequence comprising a sequence according to SEQ ID NO: 8.
- the regulatory nucleic acid is a promoter.
- the object is achieved according to the invention by a vector comprising a regulatory sequence according to the invention.
- the regulatory sequence is contained in the vector pAdCMVssTnl, the CMV promoter in this vector being replaced by the regulatory sequence according to the invention.
- the vector according to the invention is an adenoviral vector.
- the vector according to the invention comprises a coding sequence which is under the control of the regulatory nucleic acid according to the invention.
- the coding sequence is selected from the group comprising hsp70 and the "slow skeletal troponin I".
- the object is achieved by a cell comprising a regulatory nucleic acid according to the invention and / or a vector according to the invention.
- the cell is a eukaryotic cell.
- the cell is a mammalian cell.
- the cell is a human cell.
- the object is achieved according to the invention by a medicament comprising a regulatory nucleic acid according to the invention, a vector according to the invention and / or a cell according to the invention.
- the medicament is used in the context of gene therapy.
- the medicament is one for the treatment and / or prevention of cardiovascular diseases.
- cardiovascular diseases are those in which a point mutation in a gene leads to a clinical picture.
- the point mutation lies in genes which code for proteins of the sarcomere, the dystrophin and cardiac actin.
- the cardiovascular disease is selected from the group comprising hypertrophic cardiomyopathy, long QT syndrome and dilated cardiomyopathy.
- the invention is based on the surprising finding that genetic causes for the development or disposition for the development of dilated cardiomyopathy in humans are of crucial importance. This finding is based on the discovery by the inventor that variants which are associated with dilated cardiomyopathy can be found in various exons of the human MLP gene.
- the present genomic human sequence of the MLP gene is organized into six exons, all exons having the classic intron-exon transitions and elements which are characteristic of a promoter being found at the 5 ' end.
- the position of the individual exons in the human genomic sequence of the MLP gene can be described as follows:
- Exon 1 from 12983 - 1301 1 (total 28 bp)
- Exon 2 from 22480 - 22620 (total 140 bp)
- Exon 3 from 26653 - 26823 (total 170 bp)
- the entire gene thus extends to approximately 20,000 bp.
- Exon 1 of the gene encodes only a 5 ' untranslated region; the translated areas are coded on exons 2 to 6.
- a computer-aided promoter analysis showed that there is a classic TATA box in front of the 1st exon.
- a large number of patients with familial DCM were analyzed and it was surprisingly found that point mutations occur in the coding sequence of the genomic human sequence of the MLP gene, and thus also in the corresponding mRNA or the derived cDNA.
- a patient from such a family showed a base pair exchange (T-> C) in exon 2 which leads to an amino acid exchange from tryptophan to arginine at position 4 (W4R).
- the mutation causing this exchange occurs more precisely at base no. 10 of exon 2 of the translated human genomic sequence for MLP.
- the position corresponds to position 67 of the sequence according to SEQ ID No. 1.
- the mutated sequence can be found here as SEQ ID No. 2
- the variant in the MLP gene discovered by the inventor leads to a new restriction interface, which is why large patient populations can be analyzed quickly.
- a further patient collective the members of which suffer from DCM, was examined, the exon 2 being amplified from genomic DNA from patients with DCM and digested with the corresponding enzyme.
- six more patients with the variant could be detected from a collective of 500 patients.
- family trees were created and, as far as possible, family members were summoned to take blood.
- two families have so far been found in which the disease is genegosegregated.
- the amino acid tryptophan present in the wild type belongs to the heterocyclic amino acids, which is exchanged for this, ie in the O 01/57208 _-j e> _ PCT / EP01 / 01042
- Mutant nucleic acid present arginine is the most basic amino acid. So it is a structurally disruptive exchange.
- the amino acid exchange is in a highly conserved area of the protein, whereby not only the tryptophan is highly conserved at this point between the MLPs of different species (human, pig, rat, mouse), but also the neighboring amino acids at the amino terminal end of the protein. This applies not only to the MLP itself, but also to the most closely related proteins (CRPI and CRP2). It can be concluded from this that the amino-terminal region of the protein is needed to transport the MLP to its destination in the myocyte ("docking", such sequences are the same for many proteins and therefore occur ubiquitously). If so, could the modified MLP could no longer be transported to its destination and would have no biological effect.
- the variant in exon 2 has a pathogenetically important function in the genesis of a DCM in the corresponding patients with a probability bordering on certainty.
- the nucleic acids disclosed herein are important in many ways. By correlating the disclosed mutated MRL sequences with heart diseases, in particular dialatative cardiomyopathy, examinations can be carried out on biological material in order to check whether the sample and thus the person from whom the sample originates has or at least has heart disease due to the genetic makeup carries the risk of developing this heart disease.
- the disclosed nucleic acids can thus be used both for the purposes of prevention and for diagnosis.
- a patient collective can also be the subject of investigations that use the nucleic acids disclosed or make use of them.
- the sequences claimed here are not only intended to be the sequences according to SEQ ID No. 2 and SEQ ID No.
- nucleic acids disclosed herein also include those which contain the sequences according to SEQ ID No. 2 or SEQ ID No.
- each of the two sequences being separated by a nucleotide or a sequence of several nucleotides.
- An example of such a sequence is the genomic MLP sequence which has said mutations and whose coding regions are separated from one another by introns.
- genomic human MLP sequence is described in the examples herein.
- the probes disclosed herein are allele-specific oligonucleotides that are complementary to those regions of the nucleic acids that carry the mutations disclosed. These probes can be used in particular in the polymerase chain reaction as primers for the amplification of the nucleic acid and allow detection even with small sample amounts.
- the probes can be used either after the polymerase reaction or directly for the detection of the nucleic acids containing the mutations. For this purpose, the probes can be in labeled form. Such markings can include radioactive or fluorescent markers. O 01/57208 _ ⁇ g_ PCT / EP01 / 01042
- sequences of the probes or primers for detecting the mutation in exon 2 are:
- the sequence of the probes or primers for detecting the wild-type sequence corresponding to the respective mutations is:
- sample preparation steps customary for the detection of nucleic acids or gene products are used performed that are known to those skilled in the art.
- blood or biopsy material can be used as the starting material.
- the method according to the invention is based on the detection of the mutation via the generation of an additional interface for a restriction enzyme which is not present in the wild-type sequence, be it now in the genomic sequence, the mRNA or a cDNA, the nucleic acid obtained Fragments are usually separated on an agarose gel.
- an agarose gel is used, the various nucleic acid fragments can be made visible by staining with, for example, ethidium bromide or as a result of a radioactive marker.
- kits according to the invention comprise at least one of the nucleic acids, probes or a gene product of said nucleic acids.
- the kit according to the invention particularly preferably comprises at least one probe which is specific for one of the two mutations described.
- the probe corresponding to the wild type can be included as a negative control.
- the nucleic acids according to the invention can be contained in the kit as a positive and / or negative control.
- a restriction enzyme can be contained in the kit, which cuts at the interface newly created by the mutation and thus allows restriction digestion of the mutated sequence compared to the non-mutated sequence and thus to a different pattern of the - cut - Nucleic acid, ie leads to a restriction length polymorphism.
- the nucleic acids, probes or gene products can be present in a suitable buffer in the kit.
- the kit can also comprise a carrier on which a nucleic acid or a polypeptide or protein can be immobilized.
- the invention relates to a regulatory nucleic acid, in particular a promoter.
- regulatory nucleic acid The terms regulatory nucleic acid, promoter structure and promoter are used synonymously here.
- regulatory nucleic acid sequence also leads to the fact that the expression of the MLP is stress-inducible.
- the regulatory nucleic acid sequence thus represents a tissue-specific promoter structure which can also be induced by stress.
- the above-mentioned tissue specificity of the regulatory nucleic acid results from the fact that the MLP gene only occurs in muscle tissue (i.e. striated and smooth muscles); it is therefore a muscle-specific promoter.
- the gene product or the mRNA is relatively easy to detect using various techniques (eg Northern blot, PCR), which suggests a strong promoter.
- the first exon encodes only about 30 base pairs of the 5 'untranslated region. About 20-30 base pairs before the first exon there are several TATA box elements, i.e. exactly the areas to which the DNA-dependent RNA polymerase II must bind in order to copy the gene. In addition, there are several transcription initiation sequences about 10 to 20 base pairs before the first exon.
- AP-1 sequences are present immediately before the first exon. These sequences bind the so-called "leucine zipper” transcription factors, which have important functions, including in the stress inducibility of genes. The presence of these sequences underpins the stress inducibility of the MLP gene found by the inventor.
- a whole series of AP-1 sequences are found "upstream" in the first 1000 base pairs.
- a CAAT box is found around 500 base pairs upstream.
- these elements are found 80-120 base pairs in front of the gene, but an important function of this element in the promoter discovered by the inventor cannot be ruled out.
- up to about 1000 base pairs before the first exon AP-1 and CAAT sequences are found.
- the region of approximately 1000 base pairs before the first exon thus appears to be of considerable importance for the expression of the gene.
- the core region will be the area of around 500 base pairs (i.e. starting from the CAAT box) before the first exon.
- FIG. 2 The various elements of the regulatory nucleic acid disclosed herein are shown in FIG. 2.
- the regulatory sequence according to the invention can also be determined by the sequence according to SEQ ID No. 8 are shown.
- the regulatory sequence or promoter structure according to the invention can also be represented by amplifying the corresponding sequence from human genomic DNA with the aid of the polymerase chain reaction with two primers.
- the primer gP1 with the sequence is used as an upstream (“upstream”) primer
- the primer gP2 As a downstream primer, the primer gP2 with the sequence
- the regulatory sequence according to the invention can also be integrated in a vector.
- Such vectors can be both plasmid and viral vectors.
- vectors are nucleic acid molecules that serve as a recipient or carrier for — preferably foreign — nucleic acids.
- Vectors generally have an origin of replication ("origin") and genetic markers which allow the vector to be detected in host cells.
- the vector can comprise further elements which control the translation and / or transcription of the recorded or in the vector contained nucleic acid serve.
- Both the regulatory sequence according to the invention and the vector according to the invention can be contained in one cell.
- any cell or cell line can be used for this.
- the cell can be selected from the group comprising prokaryotic and eukaryotic cells.
- the cell can in turn be selected from the group which in particular comprises yeast cells, insect cells and mammalian cells.
- yeast cells yeast cells
- insect cells insect cells
- mammalian cells it can be provided that they are primary cells, primary cell cultures or established cell cultures.
- regulatory sequences there are a multitude of possible uses for the regulatory sequences and the various biological systems containing them, which are based on the following considerations and the fact that the regulatory nucleic acid disclosed herein is a tissue-specific and in particular muscle-specific expression of nucleic acid sequences under the control of said regulatory sequence allowed.
- a variety of human diseases can be attributed to genetic defects. Much of these clinical pictures, in turn, will triggered by monogenic diseases, ie mutations in a single gene. With knowledge of these molecular changes, gene therapy can repair the molecular defect.
- a number of diseases are known in the cardiovascular system in which point mutations in a single gene lead to serious clinical pictures. These include mutations in the genes that encode sarcomere proteins and lead to hypertrophic cardiomyopathy (Towbin. The role of cytoskeletal proteins in cardiomyopathies. 10, 131-139 (1998)), long QT syndrome (Jerve Lange Nielson syndrome and Romano Ward syndrome) (Neyround, N. et al .; Circ Res 84, 290-297 (1999)) and mutations in dystrophin (Muntni F. et al .; N Engl J Med 329, 921 -5 (1993)) or cardiac actin gene (Olsen, T. et al .; Science 280, 750 - 752 (1998)), which lead to dilated cardiomyopathy. In principle, these diseases can be treated causally by applying the correct gene.
- the clinical pictures listed above only affect cardiomyocytes.
- Expression of the gene to be transferred into cells other than cardiomyocytes makes no sense and only increases the dangers of gene therapy, namely the occurrence of further mutations or undesirable interactions in the sense of immune reactions.
- the regulatory nucleic acid according to the invention is only expressed in muscle tissue and therefore, and because of its strength, it is ideal for gene therapy in or of muscle tissue.
- the procedure can be such that from the shuttle vector pAdCMVssTnl, as described in Westfall et al. (Westfall, M. et al .; Proc Natl Acad Sei USA 94, 5444-5449 (1997)), with the corresponding enzyme, cut out the CMV promoter and the MLP promoter (ie a regulatory nucleic acid according to the invention) into the shuttle plasmid pAdCMVssTnl is installed.
- the resulting plasmid pAdMLPssTnl can together pJM17 via the calcium phosphate method, as in Westfall et al. (Westfall, M.
- any desired gene product can be cloned in a known manner in front of the MLP promoter in a suitable vector, for example a shuttle vector, and brought to expression.
- a suitable vector for example a shuttle vector, and brought to expression.
- the hsp70 gene which has protective functions on the myocardium, could be connected upstream (Yellon, D.
- the promoter according to the invention can be cloned in front of any desired gene product and the promoter according to the invention can thus express the nucleic acid under the control of the promoter in a tissue-specific manner with any desired virus or vector system.
- the promoter disclosed herein can be used in particular in the diseases mentioned above and disclosed herein, including in the gene therapy treatment of DCM.
- FIG 2 shows the sequence of the regulatory nucleic acid according to the invention with the promoter-specific elements.
- the human MLP cDNA was produced using the polymerase chain reaction ( : PCR) using the two primers KMLP1 and KLMP2.
- the sequence of KMLP1 is shown as SEQ ID No.11 and is as follows:
- KMLP2 The sequence of KMLP2 is shown as SEQ ID No.12 and is as follows:
- the cDNA thus obtained as the product of the PCR has a length of approximately 650 bp and contains the entire coding region of the mRNA.
- the human genomic MLP sequence was shown by screening a human genomic library using the cDNA.
- the cDNA used as a probe was radioactively marked (Feinberg, A. et al .; Anal Biochem 132, 6-13 (1983)) and hybridized with a gene bank cloned into the bacteriophage P1 (loannou, PA et al .; Nature Genetics 6 , 84-89 (1994)).
- the screening was carried out using the usual procedures known to those skilled in the art, e.g. are described in Maniatis, Fritsch & Sambrook. Molecular Cloning, Cold Spring Harbor Laboratories, (1989).
- the MLP gene itself is located on chromosome 11 p15.1 (Fung, Y.W. et al .; Genomics 28, 602-603 (1995)).
- the clone of the human genomic MLP sequence thus obtained was then sequenced using an automatic DNA sequencer.
- the positive clone comprised a total of 80,000 base pairs and contains the entire human MLP gene, which is organized into a total of six exons, as already disclosed above.
- the two mutations found by the inventor in the MLP sequence are those at base 10 of exon 2 of the translated human genomic MLP sequence, with an exchange from T to C, and one at the third position of codon 112 of exon 4 of the translated sequence, with an exchange from A to G.
- Both mutations find their correspondence in the mRNA or the cDNA derived therefrom, the position of the mutation in base 67 corresponding to the mRNA or cDNA in the case of the mutation in exon 2 and the position of the mutation in the case of the mutation in exon 4 base 393 of the mRNA or cDNA, as also shown in SEQ ID No. 1, corresponds.
- genomic DNA was isolated using a "Qiagen Blood Kit” from 1997 and a standard “Eppendorf benchtop centrifuge” as follows:
- the PCR for the amplification of exon 2 results in a PCR product with a size of 308 base pairs. If a base pair exchange from T to C has occurred in position 1 in codon 4, the restriction enzyme Nci I cuts and 2 fragments with a size of 103 base pairs and 205 base pairs are formed.
- FIG. 1 shows in more detail the result of a restriction digest of amplified exon 2 of the human genomic MLP sequence, which was applied to an agarose gel and separated.
- the two outer traces of the agarose gel each show a molecular weight standard.
- the lane labeled "wt” shows exon 2 in its wild type, with no restriction enzyme being added to the applied restriction approach.
- the lane labeled "wt + E” shows exon 2 in its wild type, with the restriction enzyme Nci I being added to the applied restriction approach has been.
- PCR products obtained were separated on an agarose gel and analyzed for their correct size. With correct sizes, which was usually the case, another part was digested with the restriction enzyme Cvi Rl at 37 ° C. overnight and separated again on an agarose gel the next morning. Regarding the incubation, it should be noted that the restriction batch could have been incubated for 30 to 60 minutes at room temperature. The overnight incubation was only for reasons of care.
- the variants or mutations in exon 2 and exon 4 of the genomic human MLP sequence or the corresponding cDNA probes can be produced.
- ASO allele-specific oligonucleotides
- SEQ ID No. 6 corresponds to:
- the melting temperature when detecting the mutation using the sequence according to SEQ ID No. 4 is 60 ° C
- the melting temperature when detecting the wild type using the sequence according to SEQ ID No. 6 is approx. 58 ° C.
- SEQ ID No. 5 corresponds to:
- SEQ ID No. 7 corresponds to:
- the melting temperature when detecting the mutation using the sequence according to SEQ ID No. 5 is 52 ° C
- the melting temperature when detecting the wild type using the sequence according to SEQ ID No. 7 is approx. 50 ° C. - o-
- exons 2 and 4 of all carriers of the mutation (s) determined by means of the restriction analysis and the probe analysis were also sequenced using standard techniques. The sequence analysis confirmed the result of the restriction and probe analysis in all cases.
- the sequencing of exons 2 or 4 or the corresponding cDNA or the corresponding cDNA of the genomic human MLP gene or the genomic MLP gene, in particular using the probes described herein can also be used as a method according to the invention for the detection of and / or screening for myocardial diseases, in particular dilated cardiomyopathy, can be used.
- the in SEQ ID No. The specified regulatory nucleic acid is shown in FIG. 2 together with the labeled promoter-specific elements. This regulatory nucleic acid is isolated using the primers gP1 and gP2 described above, corresponding to SEQ ID No. 9 and SEQ ID No. 10, possible.
- the regulatory nucleic acid sequence or the corresponding MLP promoter (1000 bp) can be amplified using the two primers mentioned.
- the AP-1 sequences are marked by underlining, with a maximum of two mismatches being accepted from the consensus sequence TGAG / CTCA.
- CAAT sequences are marked in bold, it should be noted in this regard that at the beginning of the promoter an AP-1 and a CAAT sequence overlap.
- TATA boxes are marked in bold and underlined.
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- Pharmacology & Pharmacy (AREA)
- Zoology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
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- Heart & Thoracic Surgery (AREA)
- Biotechnology (AREA)
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- Microbiology (AREA)
- Hospice & Palliative Care (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
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Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP01905718A EP1252310A2 (de) | 2000-02-03 | 2001-02-01 | Mlp-gen, nukleinsäuren, polypeptide und deren verwendung |
JP2001558022A JP2003523743A (ja) | 2000-02-03 | 2001-02-01 | Mlp遺伝子、核酸、ポリペプチド、及びその使用 |
AU2001233727A AU2001233727A1 (en) | 2000-02-03 | 2001-02-01 | Mlp gene, nucleic acids, polypeptides and the utilization thereof |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE10004857.9 | 2000-02-03 | ||
DE10004857A DE10004857A1 (de) | 2000-02-03 | 2000-02-03 | MLP-Gen, Nukleinsäuren, Polypeptide und deren Verwendung |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2001057208A2 true WO2001057208A2 (de) | 2001-08-09 |
WO2001057208A3 WO2001057208A3 (de) | 2002-07-25 |
Family
ID=7629771
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2001/001042 WO2001057208A2 (de) | 2000-02-03 | 2001-02-01 | Mlp-gen, nukleinsäuren, polypeptide und deren verwendung |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1252310A2 (de) |
JP (1) | JP2003523743A (de) |
AU (1) | AU2001233727A1 (de) |
DE (1) | DE10004857A1 (de) |
WO (1) | WO2001057208A2 (de) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5863898A (en) * | 1996-10-29 | 1999-01-26 | Incyte Pharmaceuticals, Inc. | Human lim proteins |
-
2000
- 2000-02-03 DE DE10004857A patent/DE10004857A1/de not_active Withdrawn
-
2001
- 2001-02-01 WO PCT/EP2001/001042 patent/WO2001057208A2/de not_active Application Discontinuation
- 2001-02-01 EP EP01905718A patent/EP1252310A2/de not_active Withdrawn
- 2001-02-01 AU AU2001233727A patent/AU2001233727A1/en not_active Abandoned
- 2001-02-01 JP JP2001558022A patent/JP2003523743A/ja active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5863898A (en) * | 1996-10-29 | 1999-01-26 | Incyte Pharmaceuticals, Inc. | Human lim proteins |
Non-Patent Citations (5)
Title |
---|
DATABASE EBI / EMBL [Online] 1. Februar 2000 (2000-02-01) CHEN ET AL: "A novel member of the LIM gene family involved in cardiovascular diseases" retrieved from EBI / EMBL, accession no. AF121260 Database accession no. AF121260 XP002193081 * |
DATABASE EBI / EMBL [Online] 18. Mai 1999 (1999-05-18) ZHAO ET AL: "Use of BAC end sequences from library RPCI-11 for sequence-ready map building" retrieved from EBI / EMBL, accession no. AQ530580 Database accession no. AQ530580 XP002193082 * |
DATABASE EBI / EMBL 4. Mai 1995 (1995-05-04) FUNG ET AL: "Mapping of a human LIM protein (CLP) to human chromosome 11p15.1 by fluorescence in situ hybridization" retrieved from EBI, accession no. EMBL:HS203241 Database accession no. U20324 XP002179675 * |
DATABASE EBI / EMBL 6. Januar 1999 (1999-01-06) YASUNAGA ET AL: "Cloning and characterization of the human muscle LIM protein gene" retrieved from EBI, accession no. EMBL:U72899 Database accession no. U72899 XP002179674 * |
LI ET AL: "Mutation analysis of the human muscle LIM protein in patients of familial dilated cardiomyopathy" AM. J. HUMAN GENETICS, Bd. 65, Nr. 4, Oktober 1999 (1999-10), Seite A-475 XP001031859 * |
Also Published As
Publication number | Publication date |
---|---|
EP1252310A2 (de) | 2002-10-30 |
WO2001057208A3 (de) | 2002-07-25 |
AU2001233727A1 (en) | 2001-08-14 |
DE10004857A1 (de) | 2001-08-16 |
JP2003523743A (ja) | 2003-08-12 |
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