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Neuronal serine threonine protein kinase

Neuronal serine threonine protein kinase description/claims

The present invention relates to a serine threonine protein kinase, nucleic acids that code this kinase and their use in the diagnosis and therapy of neuronal and neoplastic diseases.

Apoplexy, also known as stroke, cerebral apoplexy and apoplectic shock, is involved in about 15% of all deaths, with men and women being affected equally. Symptoms range from disturbances of consciousness to coma, and often also encompasses spastic hemiplegia, the most disparate symptoms of central-motor and sensory loss as well as focal or generalised convulsions. Every instance of apoplexy involves a circulatory disorder in the localised cerebral region that is associated with oxygen deficiency. Two basic mechanisms act as triggers. Firstly, massive bleeding, encephalorrhagia, which is involved in 15% of cases, with this generally occurring in striolenticular arteries following vascular rupture, as a result of which limited cerebral regions are destroyed. This mechanism results in a high degree of lethality. The primary disease, which came in question, is in particular hypertonia, arteriosclerosis, intracranial aneurysm and, more rarely, consumptive coagulopathy. A cerebral infarction, encephalomalacia, is involved as the second mechanism, with this being regarded as the cause in 85% of cases. A necrosis is generally formed in this connection. Causes for this include arterial thrombosis, thromboembolism or functional ischaemia associated with open vascular lumens, e.g. following a drop in blood pressure. The cerebral infarction “ischaemic necrosis” is the cause of the apoplexy in about 70-80% of cases. Arteriosclerosis often represents the underlying causal disease. The rare, slowly developing symptoms of encephalomalacia are termed “progressive stroke”. Transient symptoms of neurological loss without the formation of tissue damage (“transient ischaemic attacks”) should be considered as the early signs of a cerebral infarction. A temporary stenotically induced or microembolism-induced limited circulatory disturbance is assumed to be the cause. Diagnosis encompasses not only a general and neurological examination but also cranial computer tomography, cerebrovascular Doppler ultrasound examination, spinal tap, dynamic brain scanning, EEG and nuclear spin resonance tomography.

The molecular principles of ischaemia and the associated sequelae are to date virtually unknown. However, it may be assumed that a complex series of biochemical processes is required until apoplexy occurs.

The present invention was intended to address the technical problem of identifying genes involved in the development of apoplexy following local oxygen deficiency and thus opening up new approaches to the prophylaxis and treatment of apoplexy. The present invention was further intended to address the technical problem of identifying proteins involved in the development of apoplexy.

The said technical problems are solved by a nucleic acid that codes for a serine threonine protein kinase, with the nucleic acid being selected from: a) a nucleic acid with one of the sequences according to SEQ ID NOs. 1-4 and a nucleic acid that codes for a protein with a sequence according to one of SEQ ID NOs. 5-8; b) a nucleic acid that hybridises with a nucleic acid according to a); c) a nucleic acid which, taking account of the degeneration of the genetic code, would hybridise with a nucleic acid according to a); d) derivatives of a nucleic acid according to a)-c) that are obtained by substitution, addition, inversion and/or deletion of one or more bases; and e) a nucleic acid that is complementary to a nucleic acid according to a)-d).

For example, in derivatives of the proteins according to SEQ ID NOs. 5-8, arginine radicals are replaced by lysine radicals, valine radicals by isoleucine radicals or aspartic acid radicals by glutamic acid radicals, with the physicochemical properties of the replaced amino acid and the amino acids to be replaced being very similar (e.g. spatial filling, alkalinity, hydrophobicity). However, one or more amino acids may also be replaced within their sequence, added or removed, or several of these measures may be combined with one another. The proteins that are thus modified with respect to SEQ ID NOs. 5-8 have at least 60%, preferably at least 70% and particularly preferably at least 90% sequence identity with the sequences SEQ ID NOs. 5-8, calculated in accordance with the algorithm of Altschul et al., J. Mol. Biol., 215, 403-410, 1990. The isolated protein and its functional variants can be isolated advantageously from the brain of mammals such as Homo sapiens, Rattus norvegicus or Mus musculus. The term ‘functional variants’ should also be understood to mean homologues from other mammals.

The nucleic acids according to the invention according to SEQ ID NOs. 1-4 represent nucleic acids as isolated from the mouse (SEQ ID NOs. 1 and 2) or humans (SEQ ID NOs. 3 and 4) respectively, with the nucleic acids according to SEQ ID no. 2 and SEQ ID no. 4 being longer splice variants of SEQ ID no. 1 and SEQ ID no. 3 respectively. Nucleic acids that code for a protein that displays at least 60%, preferably at least 70% and particularly preferably at least 90% identity to one of the proteins as coded by SEQ ID NOs. 1-4 are also regarded as being in accordance with the invention.

“Derivatives” of the aforesaid nucleic acids according to the invention, e.g. allele variants, differ from the said nucleic acids according to SEQ ID NOs. 1-4 by substitution, addition, inversion and/or deletion of one or more bases, but with kinase activity being maintained. Derivatives, such as homologues or sequentially allied nucleic acid sequences, can be isolated from all mammalian species, including humans, by current methods via hybridisation with one of the nucleic acid sequences according to the invention or fragments thereof.

The term “functional equivalents” should also be understood to mean homologues of the nucleic acid according to SEQ ID NOs. 1-4, for example homologues from other mammals, shortened sequences, single-strand DNA or RNA or PNA of the coding and non-coding nucleic acid sequence. Functional equivalents of this kind can be isolated on the basis of the nucleic acids of SEQ ID NOs. 1-4, for example by standard hybridisation methods or PCR technology, from other vertebrates, such as mammals. Oligonucleotides from conserved regions that can be determined by the expert in the known way are advantageously used for hybridisation. However, longer fragments of the nucleic acids according to the invention or the entire sequence may also be used for hybridisation. Standard conditions for hybridisation vary according to the nucleic acid used—oligonucleotide, longer fragment or full sequence—or according to which nucleic acid type—DNA or RNA—is used for hybridisation. Thus, for example, melting temperatures for DNA: DNA hybrids are around 10° lower than those of DNA-RNA hybrids of the same length.

The term “standard conditions” should, for example depending on nucleic acid temperatures, be understood to mean between 42 and 58° C. in an aqueous buffer solution with a concentration of between 0.1-5×SSC (1×SSC=0.15 M NaCl, 15 mM sodium citrate, pH 7.2) or additionally in the presence of 50% formamide, such as for example 42° C. in 5×SSC, 50% formamide. Advantageously, hybridisation conditions for DNA:DNA hybrids are 0.1×SSC and temperatures between around 20° C.-45° C., preferably between around 30° C.-45° C. For DNA:RNA hybrids, hybridisation conditions are advantageously 0.1×SSC and temperatures between around 30° C.-55° C., preferably between around 45° C.-55° C. These specified temperatures for hybridisation 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 hybridisation are described in relevant genetics textbooks such as, for example, Sambrook et al., “Molecular Cloning”, Cold Spring Harbor Laboratory, 1989, and can be calculated by formulae familiar to the expert, for example depending on the length of the nucleic acids, the nature of the hybrids or the G+C content. Additional information on hybridisation can be obtained by the expert from the following textbooks: Ausubel et al. (eds), 1985, Current Protocols in Molecular Biology, John Wiley & Sons, New York; Hames and Higgins (eds), 1985, Nucleic Acids Hybridization: A Practical Approach, IRL Press at Oxford University Press, Oxford; Brown (ed), 1991, Essential Molecular Biology: A Practical Approach, IRL Press at Oxford University Press, Oxford.

The term “derivatives” of the sequences according to the invention according to SEQ ID NOs. 1-4 should also be understood to mean promoter variants upstream of the coding regions of the sequences according to the invention; these may be modified by one or more nucleotide replacements via insertion, addition and/or deletion without the promoter properties, particularly promoter strength and inducibility, being impaired. However, the term “derivative” also covers promoter variants which, based on the sequences according to the invention, are strengthened in their activity by nucleotide replacement(s).

“Neoplastic diseases” are those to do with abnormal growth behaviour of cells, the loss of intercellular inhibiting mechanisms, etc. These include, for example, carcinomas as abnormal proliferations of endodermal cells, lymphoneoplastic diseases, melanomas, etc.

A preferred nucleic acid codes for a protein with a sequence according to one of SEQ ID NOs. 5-8 or a protein that displays at least 60% identity to one of the said sequences.

In a further preferred embodiment, the nucleic acid is at least 60% identical to the coding sections of one of the sequences according to one of SEQ ID NOs. 1-4.

In a further preferred embodiment, the nucleic acid codes for a protein sequence according to one of SEQ ID NOs. 5-8, with a nucleic acid that codes for SEQ ID no. 7 being particularly preferred.

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