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WO1996017865A2 - Genes et produits geniques pour le diagnostic de lesions nerveuses degeneratives - Google Patents

Genes et produits geniques pour le diagnostic de lesions nerveuses degeneratives Download PDF

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Publication number
WO1996017865A2
WO1996017865A2 PCT/EP1995/004777 EP9504777W WO9617865A2 WO 1996017865 A2 WO1996017865 A2 WO 1996017865A2 EP 9504777 W EP9504777 W EP 9504777W WO 9617865 A2 WO9617865 A2 WO 9617865A2
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ser
gly
pro
ala
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PCT/EP1995/004777
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German (de)
English (en)
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WO1996017865A3 (fr
Inventor
Hans-Werner Müller
Clemens Gillen
Marc Gleichmann
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Boehringer Mannheim Gmbh
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Priority claimed from DE19502525A external-priority patent/DE19502525A1/de
Application filed by Boehringer Mannheim Gmbh filed Critical Boehringer Mannheim Gmbh
Priority to AU43023/96A priority Critical patent/AU693982B2/en
Priority to EP95941669A priority patent/EP0796275A2/fr
Priority to JP8517317A priority patent/JPH10511544A/ja
Publication of WO1996017865A2 publication Critical patent/WO1996017865A2/fr
Publication of WO1996017865A3 publication Critical patent/WO1996017865A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor

Definitions

  • the invention relates to new genes and gene products which are suitable for the diagnosis of nerve damage and degenerative diseases of the peripheral nerve tissue, and the use of these genes or gene products for the therapy of degenerative nerve diseases and for regeneration after traumatic nerve lesions.
  • the group of diseases that lead to nerve damage include congenital axonal and demyelinating neuropathies, toxic neuropathies (e.g. cytostatic-induced N.), motoneuropathies, ALS and inflammatory and diabetic neuropathies.
  • toxic neuropathies e.g. cytostatic-induced N.
  • the object of the invention is to provide new genes and gene products with the aid of which a diagnosis of nerve lesions and inherited neurodegenerative diseases is possible and which are suitable for the therapy of peripheral nerve damage.
  • the genes and gene products can be used to stimulate regeneration, to inhibit degenerative progression processes and / or for — in particular genetic — therapy of neurodegenerative hereditary diseases.
  • the invention relates to nucleic acids of the sequences SEQ ID NO: 1 to 30 and the amino acids of the sequences SEQ ID NO: 31-33.
  • the invention further relates to protein-coding nucleic acids which hybridize with one of the nucleic acids of the sequences SEQ ID NO: 1 to 30 in the coding region or in the 3'- or 5'-untranslated regions (standard conditions according to Sambrook et al. (8)) and code for proteins expressed in damaged and / or regenerating nerves.
  • the protein-coding nucleic acids preferably contain one of the sequences SEQ ID NO: 1-4, 7-24 in the untranslated regions.
  • SEQ ID NO 5: describes the complete cDNA of the protein encoded by clone CR 11-9 (DSM 9518).
  • SEQ ID NO: 25 the cDNA from clone NT I-2B (DSM 9515)
  • SEQ ID NO: 26 the cDNA from clone NT 1-11 (DSM 9522)
  • SEQ ID NO: 27 the cDNA from clone NT 1-12 ( DSM 9520)
  • SEQ ID NO: 28 the cDNA from clone NT 1-17 (DSM 9516).
  • a nucleic acid is preferably used which hybridizes under stringent conditions with one of the nucleic acids mentioned under standard conditions.
  • standard conditions and methods for hybridization are known to the person skilled in the art and are described, for example, by J. Sambrook et al. (1989) (8) and B.D. Harnes and S.G. Higgins (1985) (37). The standard protocols described there are usually used for this. Particular reference is made to Sambrook, Section IX, both publications being the subject of the disclosure of this invention. Standardized stringent conditions are also described in Höltke and Kessler (1990) (50).
  • Preferred stringent conditions are given for hybridization in the presence of 1 mol / 1 NaCl, 1% SDS and 10% dextran sulfate and subsequent washing of the filter twice at room temperature for 5 minutes in 2 ⁇ SSC and a further washing step for 30 minutes.
  • This further washing step can be carried out at 0.5 x SSC, 0.1% SDS, preferably at 0.2 x SSC, 0.1% SDS and particularly preferably at 0.1 x SSC, 0.1% SDS at 65 ° C become.
  • the proteins expressed in damaged and / or regenerating nerves are expediently produced recombinantly using the methods familiar to the person skilled in the art.
  • a DNA is first produced, which is able to produce such a protein.
  • DNAs are contained in the plasmids DSM 9514 - DSM 9523 and DSM 9561, which were deposited on October 25, 1994 and on November 25, 1994 (DSM 9561) with the German Collection of Microorganisms for patent purposes.
  • the sequence coding for the protein can be determined with the aid of SEQ ID NO: 1 to 30 by determining the reading frame of the cDNA clones.
  • a cDNA library preferably the cDNA library cloned into ⁇ ZAPII phages, Stratagene, US
  • nucleic acid samples in accordance with the sequences SEQ ID NO: 1 to 30 mentioned according to the invention.
  • the RNA on which the library is based preferably originates from nerve tissue (in particular from the sciatic nerve of mammals, such as rats or humans). CDNA fragments which are as close as possible to the 5 'end of the sequences should preferably be used as probes. It is also possible to use the RACE method (FINDERACE Kit, Clontech Laboratories, Darmstadt, DE) to amplify longer cDNA fragments from an RNA using a polymerase chain reaction, which can then be cloned.
  • nerve tissue in particular from the sciatic nerve of mammals, such as rats or humans.
  • CDNA fragments which are as close as possible to the 5 'end of the sequences should preferably be used as probes. It is also possible to use the RACE method (FINDERACE Kit, Clontech Laboratories, Darmstadt, DE) to amplify longer cDNA fragments from an RNA using a polymerase chain reaction, which can then be cloned.
  • the desired DNA is cloned into a vector that can be transferred to a host cell and replicable there.
  • a vector contains operator elements which are required for the expression of the DNA.
  • This vector is transferred to a host cell that is capable of expressing the DNA.
  • the host cell is cultured under conditions suitable for the amplification of the vector and the recombinant protein is obtained from the cells or the supernatant. Suitable measures familiar to the person skilled in the art ensure that the protein can assume a tertiary structure in which it is active.
  • the expressed protein it is not necessary for the expressed protein to contain precisely the sequence in the coding or in the untranslated regions which is coded by SEQ ID NO: 1 to 30. Proteins which contain essentially (preferably more than 90%) the same sequence and show similar properties are also suitable.
  • nucleic acid sequence of the gene can also be modified. Such modifications are, for example:
  • Another object of the invention is a method for producing a polypeptide, which is expressed in damaged and or regenerating nerves, by expression of an exogenous DNA in prokaryotic or eukaryotic host cells and isolation of the desired polypeptide, the DNA in the coding or untranslated characteristic of the protein Range SEQ H> NO: 1 to 30 or a DNA which codes for the same amino acid as part of the degeneration of the genetic code.
  • the protein is preferably expressed in microorganisms, in particular in prokaryotes, and there in E. coli.
  • the expression vectors must contain a promoter which allows expression of the protein in the host organism.
  • promoters are known to the person skilled in the art and are, for example, lac promoter (Chang et al. (1977) (I)), trp promoter (Goeddel et al. (1980) (2)), ⁇ p promoter (Shi atake et al. ( 1981) (3)) and T5 promoter (U.S. Patent No. 4,689,406 (4)).
  • Synthetic promoters such as tac promoter (US Pat. No. 4,551,433 (5)) are also suitable. Coupled promoter systems such as, for example, T7 RNA polymerase / promoter system (Studier et al., J. Mol. Biol.
  • Hybrid promoters comprising a bacteriophage promoter and the operator region of the microorganism are also suitable (EP-A 0267 851 (7)).
  • an effective ribosome binding site is required.
  • this ribosome binding site is referred to as the Shine-Dalgarno (SD) sequence (Sambrook et al. (1989) (8)).
  • the protein As a fusion protein.
  • a DNA which codes for the N-terminal part of an endogenous bacterial protein or another stable protein is usually attached to the 5 'end of the fused for the protein coding sequence of the invention. Examples include lacZ (Phillips and Silhavy (1990) (9)), trpE (Yansura (1990) (10)).
  • the fusion proteins are preferably cleaved with enzymes (e.g. factor Xa) (Nagai et al. (1984) (11)).
  • enzymes e.g. factor Xa
  • Further examples of the cleavage sites are the IgA protease cleavage site (WO 91/11520 (12), EP-A 0495 398 (13)) and the ubiquitin cleavage site (Miller et al. (1989) (14)).
  • the proteins expressed in this way in bacteria are obtained in a conventional manner by digesting the bacteria and isolating the proteins.
  • a fusion product is preferably used, which consists of the signal sequence which is suitable for the secretion of proteins in the host organisms used and the nucleic acid which codes for the protein.
  • the protein is either secreted into the medium (for gram-positive bacteria) or into the periplasmic space (for gram-negative bacteria).
  • a cleavage site which allows the protein to be split off either during processing or in an additional step.
  • signal sequences originate, for example, from ompA (Ghrayeb et al. (1984) (15)), phoA (Oka et al. (1985) (16)).
  • Terminators are DNA sequences that signal the end of a transcription process. They are usually characterized by two structural peculiarities: an inverted repetitive G / C-rich region that can intramolecularly form a double helix, and a number of U (or T) residues. Examples are the main terminator in the DNA of phage fd (Beck and Zink (1981) (43) and rmB (Brosius et al. (1981) (17)).
  • the expression vectors usually contain a selectable marker to select transformed cells.
  • selectable markers are, for example, the resistance genes for ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline (Davies et al. (1978) (18)).
  • suitable selectable markers are the genes for essential substances of the biosynthesis of substances necessary for the cell, e.g. Histidine, tryptophan and leucine.
  • Suitable bacterial vectors are known. For example, vectors have been described for the following bacteria: Bacillus subtilis (Palva et al. (1982) (19)), E. coli (Aman et al. (1985) (20), Studier et al. (1986) (21)) , Streptococcus cremoris (Powell et al. (1988) (22)), Streptococcus lividans and Streptomyces lividans (U.S. Patent No. 4,747,056 (23)).
  • yeast in addition to prokaryotic microorganisms, expression of the recombinant proteins according to the invention is also possible in eukaryotes (such as CHO cells, yeast or insect cells).
  • the yeast system or insect cells is preferred as the eukaryotic expression system.
  • Expression in yeast can be via three types of yeast vectors (integrating YIp (yeast integrating plasmids) vectors, replicating YRp (yeast replicon plasmids) vectors and episomal YEp (yeast episomal plasmids) vectors.
  • integrating YIp yeast integrating plasmids
  • YRp yeast replicon plasmids
  • episomal YEp yeast episomal plasmids
  • nucleic acids On the basis of the nucleic acids provided by the invention, it is possible to provide a test with which nucleic acids which encode proteins which are expressed in damaged and regenerating nerves can be detected. Such detection can take place, for example, in cells or cell lysates. Such detection can be carried out using nucleic acid diagnostics.
  • the sample to be examined is brought into contact with a probe which is selected from the group
  • nucleic acids shown in SEQ ID NO: 1 to 30 or the complementary nucleic acids are shown in SEQ ID NO: 1 to 30 or the complementary nucleic acids
  • nucleic acids which hybridize with one of the nucleic acids of a), wherein
  • the nucleic acid probe is incubated with the nucleic acid of the sample and the hybridization is optionally detected via a further binding partner of the nucleic acid of the sample and or nucleic acid probe.
  • Hybridization between the probe and nucleic acids from the sample shows the presence of the RNA of such proteins.
  • Such processes are known to the person skilled in the art and are described, for example, in WO 89/06698 (25), EP-A 0200 362 (26), USP 2915082 (27), EP-A 0 063 879 (28), EP-A 0 173 251 ( 29), EP-A 0 128 018 (30).
  • the coding nucleic acid of the sample is amplified before the test, for example using the known PCR technique.
  • a derivatized (labeled) nucleic acid probe is usually used in the context of nucleic acid diagnostics. This probe is brought into contact with a denatured DNA or RNA from the sample bound to a carrier and the temperature, ionic strength, pH value and other buffer conditions - depending on the length of the nucleic acid sample and the resulting melting temperature of the hybrid to be expected - see above chosen that the labeled DNA or RNA can bind to homologous DNA or RNA (hybridization, see also J. Mol. Biol. (1975) (31), Proc. Natl. Acad. Sei.
  • Suitable carriers are membranes or carrier materials based on nitrocellulose (eg Schleicher and Schuell, BA 85, Amersham Hybond, C), reinforced or bound powdery nitrocellulose or nylon membranes derivatized with various functional groups (eg nitro group) (eg Schleicher and Schüll, Nytran) ; NEN, Gene Screen; Amersham Hybond M .; Pall Biodyne).
  • Hybridizing DNA or RNA is then detected by incubating the support with an antibody or antibody fragment after thorough washing and saturation to prevent non-specific binding.
  • the antibody or the antibody fragment is directed against the substance incorporated in the nucleic acid probe during derivatization.
  • the antibody is marked.
  • a directly labeled DNA can also be used. After incubation with the antibodies, washing is carried out again in order to detect only specifically bound antibody conjugates. The determination is then carried out via the labeling of the antibody or antibody fragment according to methods known per se.
  • - after amplification e.g. PCR technique
  • the invention therefore comprises a method for the detection of nucleic acids which code for a protein which is expressed in damaged and / or regenerating nerves, characterized in that the sample to be examined is incubated with a nucleic acid probe which is selected from the group
  • nucleic acids shown in SEQ ID NO: 1 to 30 or the complementary nucleic acids b) nucleic acids which hybridize with one of the nucleic acids of a), wherein
  • the nucleic acid probe is incubated with the nucleic acid of the sample and the hybridization is optionally detected via a further binding partner of nucleic acid of the sample and / or nucleic acid probe.
  • nucleic acids according to the invention are therefore valuable prognostic markers in the diagnosis of nerve damage.
  • the nucleic acid of the sequence SEQ ID NO: 6 is used.
  • SEQ ID NO: 6 describes the complete sequence of NT 11-11.
  • the coding area lies between bases 362-907.
  • the corresponding protein is listed in SEQ ID NO: 31.
  • NT 13-11 is a plasmolipin cDNA extended by 234 base pairs N-terminally (Fischer and Sapirstein (1994) (44)).
  • the nucleic acid encodes a protein with a molecular weight of 19.4 kDa with four transmembrane domains.
  • NT II-11 is expressed in myelinating glial cells of the CNS (oligodendrocytes) and PNS (Schwann cells) and, like a number of other myelin proteins (PLP, PMP22, Connexin 32), is characterized by four hydrophobic transmembrane domains. Like these proteins, NT II-11 can have the property of a potential disease gene for neurodegenerative diseases. Such diseases are, for example, in the CNS and PNS, Paelizaeus-Märzbacher disease, Charcot-Marie-Tooth neuropathy type la, Pressure-Neuropathy HNPP and Dejerine-Sottas disease. NT 11-11 is a valuable marker for the detection of neurodegenerative hereditary diseases.
  • This property can be tested in an in vitro test for the growth of Schwann cells and their myelinating capacity in neuron-Schwann cell cocultures. Such a test can be carried out analogously to the test for determining the protein function of PMP22 (Zoidl et al. (1995) (45)).
  • NT II-1 SEQ ID NO: 29
  • CRII-7 SEQ ID NO: 30
  • NT II-1 shows in three domains up to 79% similarity (including conservative amino acid exchanges) to CDC4L in humans (Feuchter et al. (1992) (46)).
  • NT II-1 can be involved in regulating the cell growth of Schwann cells during nerve regeneration in order to induce the dedifferentiation of Schwann cells and to stimulate the entry of these cells into the cell division cycle.
  • the proliferation of Schwann cells is an important sub-step of nerve regeneration. Only when the division phase has ended can Schwann cells begin myelinating regenerating axons. A cell in division does not form a medullary sheath.
  • NT II-1 as a nucleic acid or protein can be a valuable therapeutic agent for the regeneration of nerve cells.
  • SEQ ID NO: 29 shows the nucleic acid sequence from NT II-l.
  • the coding region of a polypeptide with NT II-1 activity is preferably between nucleotides 1-2661 or can be extended at the N-terminal.
  • the corresponding amino acid sequence shows SEQ ID NO: 32. Partial sequences of NT II-1 are described in SEQ ID NO: 1 and SEQ ID NO: 2.
  • CRII-7 shows as nucleic acid and protein clear similarities with a macrophage-specific cysteine-rich mouse protein (MS2) and a metalloproteinase (Blobel and White (1992) (47); Weskamp and Blobel (1994) (48); Katagiri et al. (1995) (49)).
  • the glycoprotein according to the invention occurs in soluble form in the nervous system since it contains no transmembrane domain.
  • the protein also has a modulatory function in cell-cell interactions as a disintegrin. Disintegrins can change integrin-mediated interactions between cells and the extracellular matrix (ECM). Because of the two protein functions (disintegrin, metalloproteinase), a mobilizing effect on cell-cell and cell-matrix contacts can be assumed for the protein according to the invention (antithrombolytic effect, sperm-egg cell contact inhibition).
  • CRII-7 can loosen the Schwann cell ECM contacts and thus facilitate the regeneration of injured axons. It can be assumed that CRII-7 fulfills this function in particular in the early phase of the regeneration process. As a nucleic acid and protein, CRII-7 thus represents a valuable therapeutic substance for the treatment of nerve damage.
  • SEQ ID NO: 30 shows the nucleic acid sequence of CRII-7.
  • the coding region of a polypeptide with CRII-7 activity lies between nucleotides 244-2370.
  • the corresponding amino acid sequence is shown in SEQ ID NO: 33. Partial sequences of the nucleic acid sequence of CRII-7 are described in SEQ LD NO: 3 and 4.
  • CRII-7 The effect of CRII-7 can be confirmed in an in vitro test.
  • the binding of cells and matrix (fibronectin or vitronectin) in the cell culture is checked.
  • An analog test can be set up using Schwann cells and fibronectin or laminin substrates.
  • the effectiveness of NT II-1 can be tested in an in vitro model with primary Schwann cell cultures.
  • Cell proliferation can be checked by incorporating 5-bromo-2'-deoxyuridine (BrdU).
  • sequence listing either shows the complete cDNA with 5 * and 3 'untranslated region of the nucleic acids according to the invention (SEQ ID NO: 5, 6 and 25-30) or partial areas thereof.
  • Table I shows an overview. The sequences are contained in the assigned plasmids and can be determined and isolated therefrom with the aid of the sequences SEQ ID NO: 1 to 30 according to methods familiar to the person skilled in the art. In the event of deviations, the sequences contained in the plasmids are to be regarded as correct.
  • sequence is in the range consisting of 5'-, 3 * UT (untranslated
  • NT I-2B shows in a partial sequence sequence similarity to NICER elements (Cho et al.,
  • the sciatic nerve in Wistar rats is damaged by crushing (crush lesion) or cutting (transection).
  • 3 cDNA libraries were produced, starting from polyA + RNA of the undamaged sciatic nerve or the distal nerve section one week after the crush lesion (see Table II).
  • the size selection of double-stranded cDNA by Sepharose gel filtration led to the separation of small cDNA fragments ( ⁇ 400 bp).
  • the ⁇ ZAP ⁇ phage (from Stratagene) was used as the vector and can be converted into a Bluescript SK ⁇ phagemid (from Stratagene) by coinfection with an Ml 3 helper phage ("Exassist", from Stratagene).
  • the type and quantity ratio of the cDNA clones obtained after transformation represent the messenger RNAs present in the cells at that time.
  • a total of 5000 cDNA clones were transferred to replica filters in a multi-stage process.
  • a filter was hybridized with radiolabeled control nerve cDNA; the other with radioactively labeled cDNA of the regenerating nerve section one week after the crush lesion. After washing and exposure, signals appeared on the autoradiogram whose intensity was proportional to the amount of RNA of the corresponding cDNA sequence in the two tissues.
  • the clones that showed a different hybridization signal were regarded as differentially regulated, isolated and subjected to renewed hybridization (so-called "rescreen”).
  • Cross hybridization experiments in the Southern blot yielded groups of homologous clones.
  • the largest cDNA clones were sequenced and identified by computer-aided comparison (FASTA, GCG software) with previously published sequences (eg GenBankTM EMBL database).
  • the cDNA library cloned into the ⁇ ZAPII phages was plated out on agar plates (diameter: 150 mm) according to the manufacturer (Stratagene, La Jolla, CA). In order to screen the entire cDNA library with a few plates, very high phage densities of 1 - 2 x 10 5 phages / plate were chosen. The plates were incubated overnight at 37 ° C.
  • Hybond N filters After removing the Plaques of Hybond N filters (Amersham International), they are processed according to the manufacturer's instructions. The filters were prehybridized and hybridized in 7% sodium dodecyl sulfate (SDS) / 0.5 M Na phosphate pH 7.0 at 60 ° C (Church and Gilbert (1984) (33)). The hybridization is carried out with [ 32 P] dCTP-labeled samples which were produced by unidirectional PCR reaction (Sezl et al. (1991) (34)) or by random-primed labeling. The filters were washed in 2 x SSC / 0.1% SDS at 60 ° C for 15 minutes and then in 0.1 x SSC / 1% SDS at 60 ° C for 15 minutes. The exposure was at -70 ° C on a Kodak X-OMAT X-ray film.
  • the conversion into the Bluescript SK H phagemid takes place by co-infection of E.coli XLlBlue cells with an M13 exassist phage according to the manufacturer (Stratagene, La Jolla, CA).
  • the single-stranded phagemid particles contained in the supernatant are used in various dilutions for the infection of E. coli SOLR cells; the disruptive exassist phage cannot replicate in these cells.
  • Hybond N filters are applied, incubated for two hours at 37 ° C. and after being removed by microwave treatment in the bacteria are lysed in a single step, the DNA is denatured and fixed (Buluwela et al. (1989) (36)).
  • the subsequent hybridization is carried out according to the above-mentioned method.
  • the plasmid DNA was obtained using the Qiagen spin plasmid kit (Qiagen GmbH, Hilden BRD).
  • DSM 9514-DSM 9523 and DSM 9561 are available under DSM (German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg lb, D-38124 Braunschweig ) on October 25, 1994 or November 25, 1994 (DSM 9561).
  • DSM 9561 German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg lb, D-38124 Braunschweig ) on October 25, 1994 or November 25, 1994 (DSM 9561).
  • DNA fragments containing genes according to the invention or parts thereof are sequenced and appropriately modified at the ends (e.g. via PCR) and used as a whole or in combination in an expression vector for E. coli.
  • the tac promoter is used as the promoter. However, it is also possible to use other, preferably well-regulated promoters.
  • An E. coli strain that has been transformed with an expression plasmid is grown either in minimal medium or LB medium with antibiotic selection overnight at 30 or 37 ° C (e.g. in 5 ml culture).
  • the overnight culture is inoculated into a larger volume (e.g. 1 1) and allowed to continue growing. If this volume is already used for obtaining biomass, it can be induced with IPTG at an OD ⁇ O of 0.2 to 2 and the cells can continue to grow until the OD and / or the enzyme activity formed does not increase any further. At this point, centrifugation is carried out and the biomass is used to purify the proteins.
  • RNA was isolated from nerve tissue according to the method of Chomczynski and Sacchi (1987) (40)). 20 ⁇ g of total RNA were separated on a 1.2% agarose-formaldehyde gel and on nylon membranes (Amersham, Braunschweig) according to standard methods transferred (Sambrook et al. (1989) (8)). The DNA sequences SEQ ID NO: 1 to 30 were radioactively labeled as a sample (Feinberg and Vogelstein (1984), Anal. Biochem. 137, 266-267 (41)). The hybridization was carried out at 68 ° C.
  • N ⁇ I-11 complete sequence of plasmolipin
  • the proliferation rate can be determined by methods known to those skilled in the art for measuring the incorporation of e.g. 3H-thymidine or bromodeoxyuridine (BrDU) can be determined in the DNA as well as by generating and evaluating cell growth curves.
  • 3H-thymidine or bromodeoxyuridine (BrDU) can be determined in the DNA as well as by generating and evaluating cell growth curves.
  • the myelinating capacity of NTII-11 can be checked in vitro using a coculture test system.
  • the coculture system consists on the one hand of the above-mentioned transduced Schwann cells (NTII-11 overexpressing cells, NTII-1 1 underexpressing cells or control cells which carry the retroviral vector without N ⁇ I-11 cDNA sequences) and out sensory neurons that are prepared from posterior root ganglia of the embryonic rat according to the methods known to the person skilled in the art.
  • Control Schwann cells form myelin sheaths around the nerve axons within 2 weeks on collagen substrate and in culture medium containing serum, to which ascorbate and nerve growth factor are added.
  • the extent of myelin formation in the test culture can be checked by conventional light microscopic and immunocytochemical methods. In this way it can be determined whether the abnormal expression of NTÜ-11 affects the capacity of the Schwann cell for myelin formation or possibly promotes it.
  • Cell culture system primary Schwann cell culture from the rat sciatic nerve
  • Schwann cells are prepared from the sciatic nerve of 1 to 3 day old Wistar rats and immunoselected according to Brockes et al. (1979). The cultures thus obtained usually have a purity of ⁇ 99%. The almost confluent cultures are driven into growth arrest for 24 hours by adding a modified DMEM medium with the addition of 0.5% fetal calf serum (FCS) (Zoidl et al., 1995). By adding a DMEM medium with 10% FCS and 2 ⁇ M forskolin, the synchronized entry into the cell cycle is made possible. At various times after stimulation, part of the cells are subjected to the FACS analysis in order to determine the distribution in the various stages of the cell cycle. The remainder can be used to analyze NTH-1 expression and phosphorylation. The following experiments are possible.
  • FCS fetal calf serum
  • RNA is isolated according to Chomczynski and Sacchi (1987). This can be subjected to Northern blot analysis using an NTII-1-specific, radioactively labeled cDNA probe or, if the NTII-1 transcript is not very abundant, analyzed by semi-quantitative RT-PCR (Myers and Gelfand, 1991).
  • a change in the NTII-1 protein concentration can be detected by Western blot analysis using an NTH-1 specific antibody.
  • the NT ⁇ -1 protein is isolated and subjected to acidic hydrolysis.
  • the amino acid mixture obtained is analyzed by two-dimensional electrophoresis with subsequent autoradiography (Contor et al., 1987).
  • Phosphorylated tyrosine residues can be detected in a Western blot analysis with a specific antibody (Frackelton et al., 1983). After incubation of these commercially available antibodies ("py20”, ICN Biochemicals; “4G10", UBI), the antibody can be detected by means of chemiluminescence (e.g. ECL system, from Amersham).
  • phosphatases of different substrate specificity can be used. Bacterial alkaline phosphatase (Pharmacia) or acidic phosphatase from potatoes (Sigma) act on phosphorylated tyrosine, serine and threonine residues. The protein phosphatase 2A (Calbiochem) only reacts with phosphorylated serine or threonine residues, the phosphatase PTP-1B (UBI) only with phosphotyrosine residues (Anderson et al., 1990).
  • Cell culture system primary Schwann cell culture from the rat sciatic nerve
  • the specificity and effectiveness of the cell-matrix interaction could be determined by varying various parameters.
  • the extent of cell adhesion can be determined by a centrifugation experiment * after different exposure times.
  • the Schwann cells are cultivated in round bottom microtiter plates. After determining the diameter of the cell pellets and incubation with the CRII-7 protein, they are subjected to centrifugation, in which the cells are pressed together on the round bottom, so that there is a proportional relationship between the diameter of the cell pellet and the extent of the adhesion.
  • the active epitope can be determined by using CRII-7 protein fragments.
  • the tripeptide RGD (Arg-Gly-Asp) interacts with a large number of ECM molecules and is part of many disintegrin amino acid sequences.
  • Snake venom proteins such as kistrin, bitistatin or barbourin with known disintegrin activity could also be used.
  • GCCCTCCCTA CCGACAGCTC AGAGTCTGTG TCTGGGCAGA GACCGAACAC CAGTGTGGAA 120
  • MOLECULE TYPE cDNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 4:
  • GAGCCTCCAG CTTGCTTTGA TCCTGATTTA GAAAGTGTGA CTTTCCAACT TGAACTCAGC 660
  • CTACTCTTAA GCCAGCTATG CTGTGACCTA AGCCACGGCT GGGTCTTAGC ACAGGGCCAC 960
  • MOLECULE TYPE cDNA
  • SEQUENCE DESCRIPTION SEQ ID NO: 13: ⁇ TTTTKTGTCG VGAGCTMGAG GACCGAACCC AGGGCCTTGT KCTTGCTAGG 60
  • GAAAGGCTCA GACCGGGTCT TCGAATGGTT TGTGTAGGCG TTTTTATGAA GAAAATGTAA 180
  • ATCTGAGCAA CATGTTAGTG GTCCGTTACT GTAATGTCAT ACCTGAAGAA ATCGATTTAC 240
  • TCACAGCTTC TCCCTGAATG CTGCTATGGC AAGTGACCTA CTATGTAACT CTCGAGCTTG 60
  • AAAGAACATA CAACGAAAGG CTCAGACCGG GTCTTCGAAT GGTTTGTGTA GGCGTTTTTA 180

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Toxicology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
  • Neurology (AREA)
  • Cell Biology (AREA)
  • Immunology (AREA)
  • Neurosurgery (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Steroid Compounds (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne de nouveaux gènes et produits géniques, exprimés dans des nerfs périphériques dégénératifs et/ou régénératifs, ainsi que leur application à des fins diagnostiques et/ou thérapeutiques. Les acides nucléiques mentionnés dans SEQ ID NO:1 à 30 s'hybrident avec les acides nucléiques de l'invention.
PCT/EP1995/004777 1994-12-05 1995-12-05 Genes et produits geniques pour le diagnostic de lesions nerveuses degeneratives WO1996017865A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU43023/96A AU693982B2 (en) 1994-12-05 1995-12-05 Genes and gene products for use in the diagnosis of degenerative nerve damage
EP95941669A EP0796275A2 (fr) 1994-12-05 1995-12-05 Genes et produits geniques pour le diagnostic de lesions nerveuses degeneratives
JP8517317A JPH10511544A (ja) 1994-12-05 1995-12-05 変性神経損傷の診断及び再生のための新規遺伝子及び遺伝子産物

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DEP4443159.7 1994-12-05
DE4443159 1994-12-05
DE19502525.3 1995-01-27
DE19502525A DE19502525A1 (de) 1994-12-05 1995-01-27 Neue Gene und Genprodukte zur Diagnostik und Regeneration degenerativer Nervenschädigungen

Publications (2)

Publication Number Publication Date
WO1996017865A2 true WO1996017865A2 (fr) 1996-06-13
WO1996017865A3 WO1996017865A3 (fr) 1996-09-19

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1995/004777 WO1996017865A2 (fr) 1994-12-05 1995-12-05 Genes et produits geniques pour le diagnostic de lesions nerveuses degeneratives

Country Status (5)

Country Link
EP (1) EP0796275A2 (fr)
JP (1) JPH10511544A (fr)
AU (1) AU693982B2 (fr)
CA (1) CA2206994A1 (fr)
WO (1) WO1996017865A2 (fr)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL95511A (en) * 1989-08-30 2000-10-31 Max Planck Gesellschaft Neurotrophin-3 a novel neurotrophic factor related to nerve growth and brain derived neurotrophic factor
AU2010592A (en) * 1991-05-20 1992-12-30 Case Western Reserve University Neurotrophic factor, preparation and uses thereof
US5968817A (en) * 1993-03-15 1999-10-19 The Scripps Research Institute DNA encoding serotonin receptors

Also Published As

Publication number Publication date
AU693982B2 (en) 1998-07-09
CA2206994A1 (fr) 1996-06-13
JPH10511544A (ja) 1998-11-10
EP0796275A2 (fr) 1997-09-24
WO1996017865A3 (fr) 1996-09-19
AU4302396A (en) 1996-06-26

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