MXPA01005486A - A&bgr;-PEPTIDE SCREENING ASSAY - Google Patents

Abstract

The invention relates to a method for determining&ggr;-secretase activity, to individual components of the method and the application of the method. The invention provides a novel method for determining&ggr;-secretase activity and for detecting&ggr;-secretase;particular embodiments of the method relate to methods for identifying a&ggr;-secretase or a cDNA which codes for a&ggr;-secretase and methods for identifying substances which can inhibit the activity of a&ggr;-secretase. Substances of this type are particularly significant since they can be used, e.g. as pharmaceutical active agents, e.g. for treating Alzheimer's disease.

Description

PROOF OF SELECTION FOR Aß-PEPTIDO The invention relates to a process for the determination of β-secretase activity, individual components of the process and the use of the process. Alzheimer's disease is a degenerative disorder of the brain that is accompanied at the cellular level by a massive loss of neurons in the limbic system and in the cerebral cortex. In affected areas of the brain, protein deposits at the molecular level, so-called plaques, can be detected at the molecular level, which are an essential feature of Alzheimer's disease. The protein that occurs most frequently in these plates is a peptide of 40 to 42 amino acids in size which is designated as Aβ-peptide. This peptide is a product of cleavage of a significantly larger protein of 695 to 770 amino acids, the so-called precursor amyloid protein (APP). APP is an integral transmembrane protein which first crosses the lipid bilayer. By far the longest part of the protein is extracellular, while the shorter C-terminal domain is directed within the cytosol (Figure 1). The Aβ-peptide is shown dark gray in Figure 1. Approximately two-thirds of the Aβ-peptide originates from the extracellular domain and approximately one third from the transmembrane domain of APP. In addition to the membrane-based APP, a secreted form of the precursor amyloid protein consisting of the large hectodomain of APP can be detected and designated as APPsec ("secreted APP"). APPsec is formed from APP by proteolytic cleavage, which is effected by α-secretase. The proteolytic cleavage takes place at a site of the amino acid sequence of the amino acid sequence of APP that is within the amino acid sequence of the Aβ-peptide (after the amino acid residue 16 of the Aβ-peptide). The proteolysis of APP by a-secretase thus excludes the formation of the Aβ-peptide. The Aß-peptide can thus only be formed from APP in an alternative processing path. It is postulated that two additional proteases are involved in this processing route, a protease that is designated as β-secretase, unfolding in the N-terminus of the Aβ-peptide in the APP and the second protease, which is designated as β-secretase, releasing the C-terminus of the Aβ-peptide (Kang, J. et al., Nature, 325, 733) (Figure 1). Until now it has not been possible to identify any of the three secretases or proteases (a-secretase, β-secretase, β-secretase). The knowledge of secretases, however, is of great interest, particularly in the context of research on Alzheimer's disease and for the identification of the proteins involved, which can in turn be used as targets for continued studies. On the other hand, the inhibition of the β-secretase and in particular of the β-secretase could lead to a reduction in the production of Aβ, on the other hand, an activation of the α-secretase could increase the processing of APP in APPsec and thus reduce the formation of the Aß-peptide simultaneously. A transgenic C. elegans found in the course of such investigations is described in the unpublished German Patent Application which has the reference 198 49 073.9. There are many indications of the fact that Aß-peptide is a crucial factor in the presence of Alzheimer's disease. inter alia, the neurotoxicity of Aß-fibrils in cell culture is postulated (Yankner, BA et al., (1990) Proc Nat Acad Sci USA, 87 9020). In patients with Down syndrome, in which APP occurs in a copy In addition, the characteristic neuropathology of Alzheimer's disease also occurs at an age of 30 years. Here, it is assumed that over-expression of APP follows an increased conversion within the Aβ-peptide (Rumble, B. et al., (1989), N. Engl. J. Med., 320, 1446). Perhaps the strongest indication of the central role of the Aß-peptide are familial forms of Alzheimer's disease. Here, there are mutations in the APP gene around the area of cleavage sites of β- and β-secretase or in two additionally AD-associated genes (presenilins), which in cell culture lead to a significant increase in the production of Aβ (Scheuner, D. et al., (1996), Nature Medicine, 2, 864). There are a number of indications of the fact that APP is first split into the Aβ-peptide by the β-secretase during its processing to, after this, serve as a substrate for β-secretase (Maruyama, KY et al., (1994 ) Biochem, Biophys Res Commun, 202, 1517; Estus, S. et al., (1992), Science, 255, 726). The β-secretase therefore plays a crucial role in the formation of the Aβ-peptide. A demonstration of the activity of the β-secretase that is customarily used is the detection of the Aβ-peptide, which, however, frequently becomes difficult. An important reason for this is that only a small part of APP is converted to the Aβ-peptide (Simons M, et al., Neurosci (1996) 1; 16 (3): 899-908). In addition, the Aß-peptide is only a very small fragment of breakage of approximately 4 kDa and, because of its hydrophobic nature, has a great tendency for autoaggregation so that it readily precipitates under physiological conditions (Hilbich, C. et al., (1991) J. Mol. Biol., 218, 149). Detection of the Aβ-peptide in eukaryotic cells is carried out by means of immunobiological methods such as, for example, ELISA, immunoprecipitation and Western staining (Suzuki, N. et al., Science 1994, 27, 264 (5163) 1336; Haass, C. et al., (1992) Nature, 359, 322). These processes are relatively laborious, since they involve incubation with appropriate antibodies and require the destruction of the cells used, which are obtained from cell culture or model organisms (inter alia C. elegans). The present invention relates to a novel process for the determination of? -secretase activity and for the detection of? -secretase; The particular modalities of the process refer on the one hand to the processes for the identification of a? -secretase or a cDNA that codes for a? -secretase and on the other hand to processes for the identification of substances that can inhibit the activity of a ? -secretase. Such substances are of particular importance, since they can be used, for example, as active pharmaceutical compounds, for example for the treatment of Alzheimer's disease. The present invention relates to a process for the detection of β-secretase, wherein 1. a transgene is prepared and used, respectively, which encodes a fused protein and contains the following constituents: a) a first nucleotide sequence encoding for a protein containing the amino acid sequence GAIIGLMVGGVVIATVIVITLVML (SEQ ID NO: 1), b) At the 5 'end of the first nucleotide sequence, a second nucleotide sequence coding for a signal peptide, c) a promoter and, - d) if appropriate, additional nucleotide sequences that encode and / or do not code; 2. this transgene is incorporated within a cell and the fused protein is expressed; 3. The fused protein is split with the amino acid sequence SEQ ID NO: 1 by the β-secretase present in the cell, with which a first partial protein is formed, which contains the amino acid sequence GAIIGLMVGGVV (SEC DE IDENT NO 2), and a second partial protein, which contains the amino acid sequence VIVITLVML (SEQ ID NO: 3) and 4. The partial protein and / or the second partial protein are detected. invention also relates to a process for the detection of β-secretase activity, wherein 1. a transgene is prepared and used, respectively, which encodes a fused protein and contains the following constituents: a) a first nucleotide sequence that encodes for a protein containing the amino acid sequence GAIIGLMVGGVVIATVIVITLVML (SEQ ID NO: 1), b) At the 5 'end of the first nucleotide sequence, a second nucleotide sequence encoding a signal peptide, c) a promoter and, d) if appropriate, additional nucleotide sequences that encode and / or do not code. 2. this transgene is incorporated within a cell and the fused protein is expressed; 3. The fused protein is split with the amino acid sequence SEC. FROM IDENT. DO NOT. 1 for the β-secretase present in the cell, with which a first partial protein is formed, which contains the amino acid sequence GAIIGLMVGGVV (SEQ ID NO: 2), and a second partial protein, which contains VIVITLVML ( ID SECTION NO.3); 4. the amount of the second partial protein is determined and the activity of the β-secretase is determined from the amount of the second partial protein formed. The processes ("selection test for Aß-peptide", "test for? -secretase") are suitable for the in vivo detection of a? -secretase or the activity of a? -secretase, making it possible to use the processes universally, even, for example, in high performance selection ("HTS"). The processes do not have the disadvantages of the conventional detection processes mentioned above, in particular the laborious isolation and detection steps are not necessary. The basis of the process is that the C-terminal APP fragment, which is split by the? -secretase into two fragments - a first partial protein containing the amino acid sequence GAIIGLMVGGVV (SEQ ID NO: 2) and a second partial protein containing the amino acid sequence VIVITLVML (SEQ ID NO: 3), the second partial protein, which contains the amino acid sequence VIVITLVML (SEQ ID NO: 3), diffuses into the cytosol of the cell (Figure 2). This second partial protein can easily be detected in the cytosol of a cell, for example with the help of a reporter gene; it serves as a detection for a secretase range or the activity of a secretase range. The β-secretase cleavage site is located in the transmembrane domain of the APP (Kang, J. et al., (1987) Nature, 325, 733). The transmembrane domain APP has - the amino acid sequence GAIIGLMVGGVV40 IA42 TVIVITLVML. The? -secretase unfolds after V40, A42 or T43. In contrast to this, the Aβ-peptide, which is produced by eukaryotic cells in cell culture, is secreted into the supernatant medium. With the help of an adequate reporter system, the release of the second partial protein can activate the expression of a reporter protein that can be detected in eukaryotic cells. By means of the detection of the reporter protein, it can be demonstrated that a cleavage by β-secretase has taken place in the APP. As a result, the β-secretase or β-secretase activity can be determined qualitatively and / or quantitatively. The constituents of the process can be characterized in greater detail as follows: The first nucleotide sequence codes for a precursor amyloid protein (APP) or a portion thereof. Preferably, the first nucleotide sequence codes for a protein containing the amino acid sequence (SEQ ID NO: 4); SEC. FROM IDENT. DO NOT. 4 contains the SEC. FROM IDENT. DO NOT. 1. The second nucleotide sequence codes for a signal peptide, preferably for the APP signal peptide (subsequently abbreviated "SP"). The signal peptide contains, for example, the amino acid sequence SEC. FROM IDENT. DO NOT. 5. As a promoter, it is possible to use an adjustable or a constitutive promoter. The promoter may be suitable, for example, for expression in mammalian cells, in C. elegans, in yeast or in Drosophila. Suitable promoters for mammalian cells are, for example, CMV (for example: Clontech, Heidelberg, Germany), HSV TK (for example Clontech), RSV (for example Invitrogen, NV Leek, Netherlands), SV40 (for example Clontech). and LTR (for example Clontech). Promoters that can be used for C. elegans are, for example, uncll9, unc54, hspl6-2, G0A1 and sel-12. For expression in yeast, the promoters ADH1 (constitutive) (Vickova et al. (1994) Gene, 25 (5), 472-4), Gall. (Conditionally inducible) (Selleck et al. (1987) Nature 325,173-7 ), MET3 (conditional) (Cherest et al. (1987) Mol Gen Genet 210, 307-13) and Met 25 are suitable. In Drosophila, it is possible to use, for example, the promoters MT (metallothionine) (for example Invitrogen), Ac5 (Invitrogen) or Ds47 (Invitrogen). Preferably, a eukaryotic cell is used in the process, for example, a human cell or a non-human cell, for example monkey, hamster, mouse, Drosophila, zebrafish or yeast. For example, a HeLa cell, 293, H4, SH-SY5Y, H9, Cos, CHO, N2A, SL-2 or Saccharomyces cerevisiae may be employed. In a particular embodiment of the invention, a C. elegans cell is used. The cell can be a constituent of a non-human transgenic animal. In a particular embodiment, the transgenic cell can be a constituent of a transgenic C. elegans. In particular, the invention relates to processes in which yeast cells are used, for example of the strain MAV203 (Life Technologies, Rockville, MD, USA) or EGY 48 (OriGene Technologies, Inc. Rockville, MD, USA). The transgene codes for a fused protein; it is composed of the partial proteins that are encoded by the first and second nucleotide sequences and, if appropriate, additional nucleotide sequences. The fused protein thus contains the first partial protein and the second partial protein, and, if appropriate, an additional partial protein. The fused protein, for example, has the amino acid sequence SEC. FROM IDENT. DO NOT. 6. In particular, a transgene having the nucleotide sequence SEC. FROM IDENT. DO NOT. 8 can be used in the process. In particularly preferred embodiments of the process, the transgene is present in a vector. The recombinant vector may have the nucleotide sequence SEC. FROM IDENT. NO.9 This special embodiment of the invention is also referred to as an SP-ClOO-Gal 4-VP16 system. In this case, a fused protein consisting of the APP signal peptide, the ClOO fragment of APP, Gal4 and VP16 is expressed. This protein located in the transmembrane domain is split into the ClOO fragment and the second partial protein, ie the part of the fused protein that contains part of the C 100 fragment, Gal4 and VP16, is detected with the help of a reporter plasmid.

In addition to the construction transgene SPC100-Gal4-VP16, other reporter constructions are also conceivable, in which, the transcription-activation domain could be inserted between the transmembrane domain and the cytosolic domain of SPC100 or a Tag (for example MYC, FLAG) on the N- and C- term and between the transmembrane domain and the SPCIOO cytosolic domain. The nucleotide sequence that it encodes may additionally encode, for example, a protein that can be used for the detection of the second partial protein. Preferably, the nucleotide sequence that it encodes is therefore located at the 3 'end of the first nucleotide sequence. The nucleotide sequence that it encodes additionally encodes, for example, for a chimeric protein or other protein that is constructed from a number of domains, for example a protein containing a domain that binds DNA and a transcription-activation domain. In a particular embodiment of the invention, the nucleotide sequence encoding additionally encodes a protein consisting of a domain that binds Gal4 and the transcription-activation domain of VP16 (Gal4-VP16), and the additional partial protein preferably has then the amino acid sequence SEC. FROM IDENT. DO NOT. 7. In yeast cells, the additional partial protein may also contain a LexA-binding domain (eg Lex A-VP16). This additional partial protein is particularly suitable for processes in which cells of the yeast strain EGY48 are used. In particular, the invention relates to processes in which cells that are co-transfected with a reporter plasmid are used. The reporter plasmid contains a reporter gene under the control of an adjustable promoter. For example, the reporter gene can code for GFP and its derivatives, for example EGFP (Enhanced Fluorescent Green Protein), EBFP, EYFP, d2EGFP, GFPuv or Luciferase (e.g. Promega, Mannheim, Germany), CAT (e.g. Promega), SEAP (for example Clontech), ßGal (for example Clontech) or factors that induce apoptosis, for example Fas, TNF-R1, death domain and homologs (Tartaglia et al. (1993) Cell 74, 845-53), ced3, ced4, ced9. As a regulatable promoter, the reporter plasmid may contain, for example, a minimal HIV promoter, the CD4 promoter or the mec7 promoter. The selection of the appropriate regulatable promoter depends on the transcription-activation domain used. A particular embodiment of the invention relates to the implementation of the process, wherein the cells used are yeast cells. As an alternative to the yeast expression vector pDBTRP (Life Technologies Inc.) (SEQ ID NO: 11) in which the MET-25 promoter was employed in a particular embodiment (see SEQ ID NO. : 12), a large number of other expression vectors with different promoters (for example the inducible Gall promoter and the MET-25 promoter or the constitutively active ADHl promoter) and with different selection markers (ADE, LEU, TRP, HIS, LYS, PHE) can be selected. A particular embodiment is used in the use of yeast cells containing Gal4- or LexA-inducible reporter genes stably integrated in the genome or extrachromosomally present. Preferably yeast strains MaV203 (Life Technologies, Rockville, MD, U.S.A.) or EGY48 (Origene Technologies, Inc. Rockville, MD, U.S.A.) are used for these modalities. A particular embodiment of the processes refers to the use of a cell that was additionally transfected with an additional recombinant vector. Preferably, the cell that is used for these modalities does not have or hardly normally has any endogenous? -secretase or is not detectable in the activity of endogenous? -secretase [lacuna] using the aforementioned processes. This cell can be transformed using an additional vector in which a nucleotide sequence - preferably a cDNA - encoding a β-secretase is contained. For example, a cDNA library can be used. This mode of the process can then be used, inter alia, to identify a? -secretase or a cDNA that codes for a? -secretase. The cDNA libraries that can be searched for a β-secretase can be prepared from cells or tissues, for example B cells, neurons, glia cells, hippocampus, whole brain, placenta, kidney. Preferably, the cDNA is prepared from human cells or human tissues but also from other organisms (eg, hamster, rat, mouse, dog, monkey). In the case of cells without transfection that do not have activity? -secretase, but after transfection with a cDNA bank exhibit activity? -secretase, the cDNA present in the cell can code for a? -secretase. This cDNA can be isolated and verified by known processes from cells exhibiting this behavior. The invention also relates to a transgene encoding a fused protein and containing the following constituents: a) a first nucleotide sequence encoding a protein containing the amino acid sequence GAIIGLMVGGVVIATVIVITLVML (SEQ ID NO: 1), b) at the 5 'end of the first nucleotide sequence, a second nucleotide sequence coding for a signal peptide, c) a promoter and d) at least one additional nucleotide sequence at the 3' end of the first nucleotide sequence , which codes for a domain that links DNA and for a domain that activates transcription. Preferably, the first nucleotide sequence codes for APP or a portion of APP. The transgene can, for example, have the nucleotide sequence SEC. FROM IDENT. NO.8 The transgene can be present in a vector. This can, for example, have the sequence of SEC nucleotides. FROM IDENT. DO NOT. 9. The process refers to the use of a transgene and / or a vector for the production of a transgenic cell, being possible for the cell to be a constituent of a non-human organism. For example, the transgene and / or the vector can be used for the production of a transgenic C. elegans. In another particular modality, the transgene and / or the vector is used for the production of transgenic yeast cells, for example, S. cerevisiae cells. The invention also relates to a process for the production of a non-human organism, for example of a transgenic C. elegans, wherein a transgene and / or a vector containing a transgene is microinjected into the gonads of the organism, i.e. , of a C. elegans. The invention also relates to a cell that contains a transgene according to the invention and a transgenic C. elegans that contains a transgene according to the invention. The invention also relates to a cell, in particular a yeast cell, which contains a transgene according to the invention, preferably present in a suitable vector. The invention relates in particular to cells, preferably yeast cells, which can contain the transgene according to the invention and additionally a cDNA library. The invention relates to the use of transgenic or recombinant cells, preferably yeast cells, or a transgenic C. elegans in a process for the determination of? -secretase or the activity of? -secretase, the use of these cells or of a C. elegans transgenic in a process for the identification of inhibitors of β-secretase activity, and the process itself. In particular, the invention relates to processes for the identification of substances that inhibit the activity of a β-secretase, the process containing the following process steps: 1. Production of a non-human transgenic organism, for example of a C. elegans Transgenic or Saccaromyces cerensiae "or a transgenic cell, containing the transgenic non-human organism or the transgenic cell a transgene having the following constituents: a) a first nucleotide sequence encoding a protein containing the amino acid sequence GAIIGLMVGGVVIATVIVITLVML (SEC DE IDENT NO.1), b) at the 5 'end of the first nucleotide sequence, a second nucleotide sequence coding for a signal peptide and c) a promoter and the transgenic non-human organism or the transgenic cell also contains a reporter plasmid, the reporter plasmid carrying a protein-binding site, a minimal promoter and a reporter gene ero and, if appropriate, a cDNA encoding a? -secretase, wherein the transgenic non-human organism or the transgenic cell expresses the transgene and, if appropriate, the? -secretase encoded by the cDNA; 2. The transgenic non-human organism or the transgenic cell is incubated with a substance that is going to be investigated and 3. The amount of the second partial protein is detected. The invention also relates to a process for the identification of substances that inhibit β-secretase activity, wherein 1. A transgene is prepared that contains the following constituents: a) a first nucleotide sequence that encodes a protein that contains the amino acid sequence GAIIGLMVGGVVIATVIVITLVML (SEQ ID NO: 1), b) at the 5 'end of the first nucleotide sequence, a second nucleotide sequence coding for a signal peptide and c) a promoter y, d) if appropriate, additional nucleotide sequences that encode and / or do not code; 2. This transgene and a reporter plasmid and, if appropriate, a cDNA encoding a? -secretase are incorporated into a cell and the fused protein encoded by the transgene and, if appropriate, the? -secretase encoded by the CDNAs are expressed in the presence of a substance that is going to be investigated. 3. the fused protein is a) unfolds or b) does not unfold within the amino acid sequence SEC. FROM IDENT. DO NOT. 1 for the β-secretase present in the cell, as a result of which either a) a first fused protein containing the amino acid sequence GAIIGLMVGGVV (SEQ ID NO: 2) and a second partial protein containing the sequence of amino acids VIVITLVML (SEQ ID NO: 3) are formed, or b) no detectable amount of the first and / or second partial protein is formed, 4. It is determined whether a second partial protein was formed. The invention also relates to processes for the identification of substances that inhibit the activity of a? -secretase, wherein a transgene encodes a protein containing a signal peptide and SEQ. FROM IDENT.

DO NOT. 1 is expressed in the presence of a substance to be investigated and of a reporter plasmid and the effect of the substance to be investigated is determined on the amount of the second partial protein formed, the second partial protein containing the sequence of amino acids VIVITLVML (ID SECTION No. 3). The invention also relates to inhibitors of a? -secretase that are identified by the processes. The invention also relates to processes for the production of a medicament, an active pharmaceutical compound (eg, inhibitor) identified with the aid of the processes described above, further processed by means of formulation and / or mixing with a pharmaceutically tolerable carrier. The invention also relates to a test equipment for carrying out the aforementioned processes. inter alia, the processes can be used, for example, together with the C100-Gal4-VP16 system (ie a fused protein consisting of ClOO, Gal4 and VP16 or using a nucleic acid encoding a corresponding fused protein) to: Identification and determination (qualitative and / or quantitative) of the activity of a? -secretase. 2. Identification of? -secretases in different tissues, cells and organism or species. Identification and isolation of the relevant cDNAs coding for this β-secretase and the additional use of the cDNAs. 3. Selection in vivo, for example yeast cells (Saccharomyces cereviriae) or in C. elegans, being possible to determine the activity of the β-secretase without the use of immunobiological methods. 4. Use of processes for the identification and characterization of substances, for example active pharmacological compounds, that modulate the activity of β-secretase, for example β-secretase inhibitors. In particular, this process can be used in an HTS (High Performance Selection). For example, substances that can be used for the treatment of Alzheimer's disease and / or for preventive treatment can be identified. 5. Investigations in the context of Alzheimer's disease, for example with mutated APP or ClOO. 6. The fused proteins / transgenes described, for example ClOO in SP-C100-Gal4-VP16, can be replaced by complete APP and β-secretase, their activity and regulation can be investigated in the same way with the aid of the processes. Figure 1: Figure 1 shows the precursor amyloid protein (Isoform APP695 and Isoforms APP770 or APP 751) and secretase cleavage products. Figure 2: Schematically shows the principle on which the processes are based: cleavage site β-secretase in the N-term; cleavage site of? -secretase in the transmembrane domain; C100 = ClOO fragment of APP; Gal4-VP16 = DNA-binding domain, a domain that activates transcription (which consists of a domain that binds DNA and a transcription activator), which binds to the domain that binds protein on the DNA of the reporter plasmid. Figure 3: Construction of the expression plasmids SP-C100-Gal4-VP16: aa = amino acids; cleavage restriction sites Sac I, Hind III and Kpn I indicating the position of the cleavage site on the plasmid. Figure 4: Expression of the plasmid pDBTRP-Met25-SP-C100-Gal4-VPl6: Construction of the expression plasmid for the expression of the transgene in yeast.

Examples: Example 1: Construction of the expression plasmid SP-C100- Gal4-VP16 The plasmid encodes the APP (SP) signal peptide which is fused to the 100 C-terminal amino acid residues of APP (ClOO). ClOO begins with the N-terminus of the Aß-peptide and ends with the C-terminus of APP. It must be further expanded by the? -secretase to release the Aß-peptide. Gal4-VP16 was fused to the C-terminus of SP-C100. Gal4-VP16 is composed of the first 147 amino acid residues of the yeast transcription activator Gal4 and the 78 C-terminal amino acid residues of VP16, a transcriptional activator of the herpes simplex virus. As a fused protein, the Gal4 fragment takes over the function of the DNA binding while the VP16 fragment activates the transcription (Sadowski et al., (1998) Science 335, 563). pcDNA3.1 + from Introgen, Netherlands, serves as a plasmid vector. Example 2: Construction of reporter plasmid pGL2 MRG5 EGFP Reporter plasmid pGL2 MRG5 has five sites that bind Gal4 forward of the HIV-TATA box. For easier detection in cell culture, the luciferase gene was exchanged for the EGFP (Enhanced Fluorescent Green Protein) gene from the pEGFP NI vector from Clontech, Heidelberg.

Example 3: Human neuroblastoma cells (SH-SY5Y cells) were co-transfected with both plasmids and then analyzed microscopically under irradiation with light of wavelength of 480 nm by means of which EGFP was excited. In some cases, it was possible to detect luminescent cells with a strong green color. Since this effect could also be based on the expression of EGFP by the reporter plasmid without specific activation, SH-SY5Y cells were transfected only with reporter plasmid. In these cells, no green fluorescence was detected. The expression must therefore be activated by Gal4-VP16, which presupposes a proteolytic release of the APP-C-term. Until now, apart from? -secretase, no additional proteolytic activities have been described that process proteolytically APP within the transmembrane domain or in the cytoplasmic part. It is therefore assumed that the release of the APP-C-term, fused to Gal-VP16, is based on the activity of the β-secretase.

Example 4: Use of the C100-Gal4-VP16 system for the detection of a cDNA encoding a β-secretase activity in cDNA libraries: SPC100-Gal4-VP16 was cloned into the yeast expression vector pDBTRP (Life Technologies Rockville, MD, USA) under the control of the MET25 promoter and the MaV203 yeast strain (Life Technologies) was transformed using these constructions. Yeast strain Ma V203 is genetically modified and contains three Gal4 inducible reporter genes (URA3, HIS3, LacZ), which are stably integrated into the genome. The expression of SPC100-Gal4-VP16 cDNA in MaV203 allowed only a small activity of reporters, such that this system is suitable for a search for a? -secretase in a cDNA library.

Example 5: The recombinant MaV203 cells of Example 4 can be used, for example, for the identification of? -secretases or selection of a human B-cell cDNA library (American Type Culture Collection, Manassas, VA, U.S.A.).

Analogously, a human hippocampal cDNA library, integrated within the yeast expression vectors p415-MET25 (ATCC, Nucleic Acid Research, 1994, Vol. 22, No. 25.5767) or p415-ADH1 (ATCC, GENE, 1995, 158: 119-122), could also be used to select a cDNA coding for a? secretase or a protein that has a? -secretase activity.

(ID SECTION NO .: 1) GAI IGLMVGGWIATVIVITLVML First partial protein (SEQ ID NO: 2) GAIIGLMVGGVV Second partial protein (SEQ ID NO: 3) VIVITLVML Amino acid sequence of a C100 fragment (SEQ ID NO: 4) LDAEFRHDSG YEVHHQKLVF FAEDVGSNKG AIIGL VGGV VIATVIVITL VMLKKKQYTS IHHGVVEVDA AVTPEERHLS KMQQNGYENP TYKFFEQMQN Amino acid sequence of a APP signal peptide (SEQ ID NO: 5) MLPGLALFLL AA TARA Amino acid sequence of a SP-C100 fragment (SEQ ID NO: 6) MLPGLALFLL AA TARALDA EFRHDSGYEV HHQKLVFFAE DVGSNKGAI I GLMVGGVVIA TVIVITLVML KKKQYTS IHH GWEVDAAVT PEERHLSKMQ QNGYENPTYK FFEQMQN Domain that activates VP16 transcription with domain that binds Gal4 (Gal4-VP16) (SEQ ID NO: 7) MKLLSSIEQA CDICRLKKLK CSKEKPKCAK C KNNWECRY SPKTKRSP T RAHLTEVESR LERLEQLFLL IFPREDLDMI LKMDSLQDIK ALLTGLFVQD NVNKDAVTDR LASVETDMPL TLRQHRISAT SSSEESSNKG QRQLTVSPEF PGI APPTDV SLGDELHLDG EDVAMAHADA LDDFD DMLG DGDSPGPGFT PHDSAPYGAL D ADFEFEQM FTDALGIDEY GG Nucleotide sequence of a transgene encoding SP-C100-Gal4-VP16 (SEQ ID NO: 8) G CAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTAC GGGCC? GATATACGCGTTGACATTGATTATTGACTAGTTATTAA7AGTAATCAATTACGGGGTCATTAGTTC ATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACC CCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAAT GGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTA TTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTT GGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTG GATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTG? TTTGGCACC GCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCAC7GCTTACTGGCTTATCGA AATTAATACGACTC? CTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTCACAGCTAGCGCA CTCGGTGCCCCGCGCAGGGTCGCGATGCTGCCCGGTTTGGCACTGTTCCTGCTGGCCGCCTGG? CGGCTCGGGCG CTGGATGCAGAATTCCGACATGACTCAGGATATGAAGTTCATCATC.V? IATTGGTGTTCTTTGCAGAAGATGTG GGTTCAAACAAAGGTGCAATCATTGGACTCATGGTGGGCGGTGTTGTCATAGCGACAGTGATCGTCATCACCTTG GTGATGCTGAAGAAGAAACAGTACACATCCATTCATCATGGTGTGGTGGAGGTTGACGCCGCTGTCACCCCAGAG GAGCGCCACCTGTCCAAGATGCAGCAGAACGGCTACGAAAATCCAACCTACAAGTTCTTTGAGCAGATGCAGAAC GCGCGGGGTACCCCGGCG ATGAAGC TACTGTCTTC TATCGAACAA GCATGCGATA TTTGCCGACT TAAAAAGCTC AAGTGCTCCA AAGAAAAACC GAAGTGCGCC AAGTGTCTGA AGAACAACTG GGAGTGTCGC TACTCTCCCA AAACCAAAAG • GTCTCCGCTG ACTAGGGCAC ATCTGACAGA AGTGGAATCA AGGCTAGAAA GACTGGAACA GCTATTTCTA CTGATTTTTC CTCGAGAAGA CCTTGACATG ATTTTGAAAA TGGATTCTTT ACAGGATATA AAAGCATTGT TAACAGGATT ATTTGTACAA GATAATGTGA ATAAAGATGC CGTCACAGAT AGATTGGCTT CAGTGGAGAC TGATATGCCT CTAACATTGA GACAGCATAG AATAAGTGCG ACATCATCAT CGGAAGAGAG TAGTAACAAA GGTCAAAGAC AGTTGACTGT ATCG CCGGAATTCCCGGGGATCTGGGC CCCCCCGAC CGATGTCAGC CTGGGGGACG AGCTCCACTT AGACGGCGAG GACGTGGCGA TGGCGCATGC CGACGCGCTA GACGATTTCG ATCTGGACAT GTTGGGGGAC GGGGATTCCC CGGGGCCGGG ATTTACCCCC CACGACTCCG CCCCCTACGG CGCTCTGGAT A GGCCGACT TCGAG7TTGA GCAGATGTTT ACCGATGCCC TTGGAATTGA CGAGTACGGT GGGTAG Nucleotide sequence of the mammalian expression vector pcDNA3.1 + (Invitrogen, Netherlands (SEQ ID NO: 9) GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTA? GCCAG7ATCTGCTCCC7GCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAA GGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTAC GGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCA? TTACGGGGTCATTAGT C "TAGCCCATATA7GGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACC CCCGCCCAT7GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCAT7GACGTCAAT GGG7GGACTATTTACGGTAAAC7GCCCACTTGGCAG7ACATCAAGTGTATCATATGCCAAGTACGCCCCCTA TTGACGTCAA7GACGGTAAATGGCCCGCCTGGCATTATGCCCAG7ACATGACCTTATGGGACTTTCCTACTT GGCAGTACATCTACG7AT? AGTCATCGCTATTACCATGGTGATGCGG7TTTGGCAGTACATCAATGGGCGTG GA7AGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGT7TGTTTTGGCACC AAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCA7TGACGCAAATGGGCGGTAGGCGTGTAC GGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAAT7A ATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGG7ACCGAGCTCGGATCCAC TAGTCCAG7GTGGTGGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGG3CCCGTTTAA ACCCGCTGA7CAGCC7CGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGT TTGCCCCTCCCCCGTGCCTTCC TTGACCCTGGAAGGTGCCACTCCCACTG7CCTTTCCTAATAAAATGAGGAAAT7GCATCGCA7TGTCTGAG7? GGTGTCAT7CTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGAT7CGGAAGACAATAGCAGG CATGCTGGGGATGCGGTGGGCTCTA7GGCTTCTGAGGCGGAAAGAA.CCAGCTGGGGCTCTAGGGGG7A7CCC CACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCC AGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCC7TTCTCGCCACGTTCGCCGGCTTTCCCCGTCAA GCTCTAAATCGGGGCATCCCTTT? GGGTTCCGATTTAGTGCT7TACGGCACCTCGACCCCAAAAAACTTGAT TAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGG7TTTTCGCCCTTTGACGTTGGAGTCCACG TTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTA7CTCGGTCTATTCTTTTGAT7TA TAAGGGATTTTGGGGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATT7A ».CGCGAATTAA TTCTGTGGAATGTG7GTCAGT7AGGGTGTGGAAAGTCCCCAGGCTCCCCAGGCAGGCAGAAGTATGCAAAGC? TGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGC ATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGT TCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTAT7TATGCAGAGGCCGAGGCCGCCTCTGCCTCT GAGCTA7TCGAGAAGTAGTGAGGAG GCTTTT7TGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCT7GT ATATCCATTTTCGGA7CTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCAC GCAGGXTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTA7GAC7GGGCACAACAGACAATCGGCTGCTCT GATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCC CTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGC? CCTGTG CTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCA TCTCACCTTGC7CCTGCCGAGAAAGTA7CCATCATGGCTGATGCAATGCGGCGGCTGCATACGC7TGATCCG GC7ACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGG7CTT GTCGATC? GGATGATCTGGACGAAGAGCATCAGGGGC7CGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCG CGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATCCCTGCTTGCCGAATATCATGGTGGAAAAT GGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCT ACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCT7CCTCGTGC1TTACGG7ATCGCCGCT CCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTG? GCGGG? CTCTGGGGTTCGAAA TGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCC77CTATGAAAGGTTGG GCTTCGGAA7CGTrTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTC7TCG CCCACCCCA? CTTG7T7ATTGCAGCTTATAATGGTTACAAATAAA.GCAATAGCATCACAAATTTCACAAATA AAGCATTTT7TTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATC? ATGTATCTTATCATGTC7GTATAC CG7CG71CCTCT? GCT? GAGCTTGGCGTAATCATGGTCATAGC7GTT7CCTG7GTGAAATTGTT? TCCGCTCA CAATTCCACAC.AZICA ^ ACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCT.V GAGTGAGCTAACTCA CATTAJVTTGCGTTGCGC7CACTGCCCGCTT7CCAGTCGGGAAACCT GTCGTGCCAGCTGCA7T? ATGAATCG GCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGñCTCGCTGCGCT CGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGG7AATACGGTTATCCACAGAA7CAGGGG? TAACGC? GGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGG CGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACC CGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGG? AGCTCCCTCGTGCGCTCTCCTGTTCCGAC CTGC CGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGT ATC7CAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACG? ACCCCCCGTTCAGCCCGACCGCT GCGCCTT? TCCGGTAACTATCGTCTTGAGTCCAACCCGGTA ^ GAC? CGACTTATCGCCAC7GGCAGC? GCGA CTGG7AACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGC7AC? GAGTTC7TGAAGTGGTGGCCTAACTACG GC ACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAG7TACCTTCCGAAAAAGAG7 '; 'GGTA GCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTG7TTGCAAGCAGCAGAT ACGCGCA GAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCAC GTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTT TTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTA TCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGG AGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAG CAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTA TTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTA C? GGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAG TTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGT TGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGAT GCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTC7T GCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTT CTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCA ACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAA AAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTT ATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGC GC? CATTTCCCCGAAAAGTGCCACCTGACGTC Nucleotide sequence of APP (SEQ ID NO: 10) AGTTTCCTCG GCAGCGGTAG GCGAGAGCAC GCGGAGGAGC GTGCGCGGGG GCCCCGGGAG ACGGCGGCGG TGGCGGCGCG GGCAGAGCAA GGACGCGGCG GATCCCACTC GCACAGCAGC GCACTCGGTG CCCCGCGCAG GGTCGCGATG CTGCCCGGTT TGGCACTGCT CCTGCTGGCC GCCTGGACGG CTCGGGCGCT GGAGGTACCC ACTGATGGTA ATGCTGGCCT GCTGGCTGAA CCCCAGATTG CCATGTTCTG TGGCAGACTG AACATGCACA TGAATGTCCA GAATGGGAAG TGGGATTCAG ATCCATCAGG GACCAAAACC TGCATTGATA CCAAGGAAGG CATCC7GCAG TATTGCCAAG AAGTCTACCC TGAACTGCAG ATCACCAATG TGGTAGAAGC CAACCAACCA GTGACCATCC AGAACTGGTG CAAGCGGGGC CGCAAGCAGT GCAAGACCCA TCCCCACTTT GTGATTCCCT ACCGCTGCTT AGTTGGTGAG TTTGTAAGTG ATGCCCTTCT CGTTCCTGAC AAGTGCAAAT TCTTACACCA GGAGAGGATG GATGTTTGCG AAACTCATCT TCACTGGCAC ACCGTCGCCA AAGAGACATG CAGTGAGAAG AGTACCAACT TGCATGACTA CGGCATGTTG CTGCCCTGCG GAATTGACAA GTTCCGAGGG GTAGAGTTTG TGTGTTGCCC ACTGGCTGAA GAAAGTGACA ATGTGGATTC TGCTGATGCG GAGGAGGATG ACTCGGATGT CTGGTGGGGC GGAGCAGACA CAGACTATGC AGATGGGAGT GAAGACAAAG TAGTAGAAGT AGCAGAGGAG GAAGAAGTGG CTGAGGTGGA AGAAGAAGAA GCCGATGATG ACGAGGACGA TGAGGATGGT GA7GAGGTAG AGGAAGAGGC TGAGGAACCC TACGAAGAAG CCACAGAGAG AACCACCAGC ATTGCCACCA CCACCACCAC CACCACAGAG TCTGTGGAAG AGGTGGTTCG AGTTCCTACA ACAGCAGCCA GTACCCCTGA TGCCGTTGAC AAGTATCTCG AGACACCTGG GGA7GAGAAT GAACATGCCC ATTTCCAGAA AGCCAAAGAG AGGCTTGAGG CCAAGCACCG AGAG? GAATG TCCCAGGTCA 7GAGAGAATG GGAAGAGGCA GAACGTCAAG CAAAGAACTT GCCTAAAGCT GATAAGAAGG CAGTTATCCA GCATTTCCAG GAGAAAGTGG AATCTTTGGA ACAGGAAGCA GCCAACGAGA GACAGCAGCT GGTGGAGACA CACATGGCCA GAGTGGAAGC CATGCTCAAT GACCGCCGCC GCCTGGCCCT GGAGAACTAC ATCACCGCTC TGCAGGCTGT TCCTCCTCGG CCTCGTCACG TGTTCAATAT GCTAAAGAAG TATGTCCGCG CAGAACAGAA GGACAGACAG CACACCCTAA AGCATTTCGA GCATGTGCGC ATGGTGGATC CCAAGAAAGC CGCTCAGATC CCCTCCCAGG TTATGACACA CCTCCGTGTG ATTTATGAGC GCATGAATCA GTCTCTCTCC CTGCTCTAC? ACGTGCCTGC AGTGGCCGAG GAGATTCAGG ATGAAGTTGA TGAGCTGCTT CAGAAAGAGC AAAAC7ATTC AGATGACGTC TTGGCCAACA TGATTAGTGA ACCAAGGATC AGTTACGGAA ACGATGCTCT CATGCCATCT TTGACCGAAA CGA? AACCAC CGTGGAGCTC CTTCCCGTGA ATGGAGAGTT CAGCCTGGAC GATCTCCAGC CGTGGCATTC TTTTGGGGCT GACTCTGTGC CAGCCAACAC AGAAAACGAA GTTGAGCCTG 7TTGATGCCCG CCCTGCTGCC GACCGAGGAC TGACCACTCG ACCAGGTTCT GGGTTGACAA ATATCAAGAC GGAGGAGATC TCTGAAGTGA AGATGGATGC AGAATTCCGA CATGACTCAG GATATGAAGT TCATCATCAA TCTTTGCAGA A ATTGGTCT AGATGTGGGT TCAAACAAAG GTGCATCAT TGGACTCATG G7GGGCGGTC TTGTCATAGC GACAGTGATC GTCATCACCT TGGTGATGCT GAAGAAGAAA CAGTACACAT CCATTCATCA TGGTGTGGTG GAGGTTGACG CCGCTGTCAC CCCAGAGGAG CGCCACCTGT CCAAGA7GCA GCAGAACGGC TACGAAAATC CAACC? ACA¡ GTTCTTTGAG CAGATGCAGA ACTAGACCCC CGCCACAGCA GCCTCTGAAG TTGGACAGCA AAACCATTGC T7CACTACCC ATCGGTGTCC ATTTATAGAA TAATGTGGGA AGAAACAAAC CCGTTTTATG ATTTACTCA7 TATCGCCTTT TGACAGCTGT GCTGTAACAC AAGTAGATGC CTGAACTTGA ATTAATCCAC ACATCAGTAA TGTATTCTAT CTCTCTTTAC ATTTTGGTCT CTATACTACA T7ATTAATGG GTTTTGTGT A CTGTAAAGAA TTTAGCTGTA TCAAACTAGT GC TGAATAG A7TCTCTCCC GATTAT7TAT CACATAGCCC CTTAGCCAGT TGTATATTA? TCTTGTGGTT TGTGACCCAA TTAAGTCCTA CTTTACATAT GCTTTAAGAA TCGATGGGGG ATGCTTCATG TGAACGTGGG AGTTCAGCTG CTTCTCTTGC CTAAGTATTC CTTTCCTGAT CACTATGCAT TTTAAAGTTA AACATTTTTA AGTATTTCAG ATGCTTTAGA GAG? TTTTT7 TTCCATGACT GCATTTTACT GTACAGATTG CTGCTTCTGC TATATTTGTG ATATAGGAAT TAAGAGGATA CACACGTTTG TTTCTTCGTG CCTGTTTTAT GTGCACACAT TAGGCATTGA GACTTCJAAGC TTTTCTTTTT TTGTCCACGT ATCTTTGGGT CTT7GATAAA GAAAAGAATC CC7GTTCATT CTAAGC? CTT TTACGGGGCG GGTGGGGAGG GGTGCTCTGC TGGTCTTCAA TTACCAAGAA TTCTCCAAAA CAATTTTCTG C ? GGATGATT GTACAGAATC ATTGCTTATG ACATGATCGC TTTCTACACT GTATTACATA AATAAATTAA ATAAAATAAC CCCGGGCAAG ACTTTTCTTT GAAGGATGAC TACAGACATT AAATAATCGA AGTAATTTTG GGTGGGGAGA AGAGGCAGAT TCAATTTTCT TTAACCAGTC TGAAGTTTCA TTTATGATAC AAAAGAAGAT GAAAATGGAA GTGGCAATAT AAGGGGATGA GGAAGGCATG CCTGGACAAA CCCTTCTTTT AAGATG7GTC 7TCAATTTGT ATAAAATGGT GTTTTCATGT AAATAAATAC ATTCTTGGAG GAGC Nucleotide sequence of plasmid pDBTRP (SEQ ID NO: 11) GACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTT7CTTAGGACGGATCGC TTGCCTGTAACTTACACGCGCCTCGTATCTTTTAATGATGGAATAATTTGGGAATTTACTCTGTGTTTATTTATT TTTATGTTTTGTATTTGGATTTTAGAAAGTAAATAAAGAAGGTAGAAGAGTTACGGAATGAAGAAAAAAAAATAA ACAAAGGTTTAAAAAATTTCAACAAAAAGCGTACTTTACA7ATATATTTATTAGACAAGAAAAGCAGATTAAATA GATATACATTCGATTAACGATAAGTAAAATGTAAAATCACAGGATTTTCGTGTGTGGTCTTCT? CACAGACAAGA TGAAACAATTCGGCATTAATACCTGAGAGCAGGAAGAGCAAGAT? AAAGGTAGTATTTGTTGGCGA7C0CCCTAG AG7CTTTTACATCTTCGGAAAACAAAAACTATTTTTTCTTTAATTTCTTTTTTTACTTTCTATTTT7AATTTATA TATTTATATTAAAAAATTTAAATTATAATTATTTTTATAGCACGTGATGAAAAGGACCCAGGTGGCACTTTTCGG GGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAA CCCTGATA / iATGCTTCAATAATCTGCAGCTCTGGCCCGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAA. • V-A7ATATCATCATGAACAATAAAACTGTC7GCTTACATAAACAGTAATACAAGGGGTGTT? TG? GCC? TATTCA ACGGGAAACGTCTTGCTGGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGG7ATAAATGGGCTCG CGATAATGTCGGGCAATCAGGTGCGACAATCTTTCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAA ACATGGC AAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCC TCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCGCGGGAAAAC AGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCG GTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACG AATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAA AGAAATGCATACCCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTC? CTTGATAACCTTAT TTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGA7ACCAGGATCTTGC CATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAA TCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTT7TTCTAATCACAATTGGTT ?? TTGGTTGTA AC? CTGGCAGAGCATTACGCTGACTTGACGGGACGGCGCATGACCAAAATCCCTTAACGTGAGTT "TCGTTCCAC TGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTG C? AACAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATC? AGAGCTACCAACTCTTTTTCCGAAGGTA? CTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTT? GGCCACCACTTCAAGAAC TCTGTAGCACCGCCTACATA CCTCGCTCTGCTAATCCTGTTACC? GTGGCTGCTGCCAGTGGCGATAAGTCGTGT CTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACA CAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTT CCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCA GGGGGGAACGCC7GGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGC TCGTCAGGGGGGCCGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCT TTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTG? GTGAGCTGAT ACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAA C GCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGT GAGCGCAACGCAATTAATGTGAGTTACCTCACTCATT? GGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCCT ATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGGA A7TAACCCTCACTAAAGGGAACAAAAGCTGGTACCGATCCCGAGCTTTGCAAATTAAAGCCTTCGAGCG7CCCAA AACCTTCTCAAGCAAGGTTTTCAGTATAATGTTACATGCGTACACGCGTCTGTACAGAAAAAAAAGAAAAAT7TG AAATATAAATAACGTTCTTAATACTAACATAA CTATAAAAAAATAAA7AGGGACC7AGACTTCAGGTTGTCTAAC TCCTTCCTTTTCGGT7AGAGCGGATGTGGGGGGAGGGCGTGAATGTAAGCGTGACATAACTAATTACATGATATC GACAAAGGAAAAGGGGCCTGTTTACTCACAGGCTTTTTTCAAGTAGGTAATTAAGTCGTTTCTGTCTTTTTCC7T CTTCAACCCACCAA? GGCCATCTTGGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT7TTTTTT7TTTTT TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCATAGAAATAA7? CAGAAGTAGATGTT GAAT7AGATTAAACTGAAGATATATAATTTATTGGAAAATACATAGAGCTTTT7GT7GATGCGCT7AAGCGATCA ATTCAACAACACCACCAGCAGCTCTGATTTTTTCTTCAGCCAACTTGGAGACGAATCTAGCTTTGACGATAACTG GAACAT7TGGAA7TCTACCCTTACCCAAGATCTTACCGTAACCGGCTGCCAAAGTGTCAA? AACTGGAGCAGTTT CCTTAGAAGCAGATTTCAAGTATTGGTCTCTCTTGTCTTCTGGGATCAATGTCCACAATTTGTCCAAGTTCAAGA C7GGCTTCCAGAAATGAGCTTG7TGCTTGTGGAAGTATCTCATACCA ^ CCTTACCGAPATAACCTGGATGGTATT TATCCATGTTAATTCTGTGGTGATGTTGACCACCGGCCATACCTCTACCACCGGGGTGCTTTCTGTGCTTACCGA TACGACCTT7ACCGGCTGAGACGTGACCTCTGTGCTTTCTAGTCTTAGTGAATCTGGAAGGCATTCTTGATTAGT TGGATGATTGTTCTGGGATTTAATGCAAAAATCACTTAAGAAGGAAAATCAACGGAGAAAGCAAACGCCATCT7? AATATACGGGATACAGATGAAAGGGTTTGAACCTATCTGGAAAATAGCATTAAACAAGCGAAAAACTGCGAGGAA AATTGTTTGCGTCTCTGCGGGCTATTCACGCGCCAGAGGAAAA7AGGAAAAATAACAGGGCA7TAGAAAAATAAT TTTGATTTTGGTAATGTGTGGGTCCTGGTGTACAGATGTTAC? TTGGTTACAGTACTCTTGT7TTTGCTGTGTTT TTCGATGAATCTCCAAAATGGTTGTTAGCACATGGAAGAGTCACCGATGCTAAGTT? TCTCT? TGTAAGCTACGT GGCGTGACTTTTGATG? AGCCGCACAAGAGATACAGGATTGGCAACTGCAAATAGAATCTCGGGATCCCCCCTCG AGATCCGGGATCGAAGAAATGATGGTAAATGAAATAGGAAATC ?? GGAGCATGAAGGCAAAAGACAAATATAAGG GTCGAACGAAAAATAAAGTGAAAAGTGTTGATATGATGTATTTGGCTTTGCGGCGCCG? AAAAACGAGTTTACGC AATTGCACAATCATGCTGACTCTGTGGCGGACCCGCGCTCTTGCCGGCCCGGCGATACGCTGGGCGTGAGCCTG TGCCCGGCGGAGT7TTTTGCGCCTGCATTTTCCAAGGTTTACCCTGCGCTAAGGGGCGAGAT7GGAGAAGCAATA AGAATGCCGGTTGGGGTTGCGATGATGACGACCACGACAACTGG7GTCATTATTTAAGTTGCCGAAAGAACCTGA G7GCATT7GCAACATGAGTATACTAGAAGAATGAGCCAAGAC7TSCGAGACGCGAGTTTGCCGGTGGTGCGAACA ATAGAGCGACCATGACCTTGAAGGTGAGÁCGCGCATAACCGCTAGAGTACTTTGAAGAGGAAACAGCAATAGGGT TGCTACCAGTATAAATAGACAGGTACATACAACACTGGAAATGGTTGTCTGTTTGAGTACGCTTTCAATTCATTT GGGTGTGCACTT TATTATGTTACAATATGGAAGGGAACTTTACACTTCTCCTATGCACATATATTAATTAAAGTC CAATGCTAGTAGAGAAGGGGGGTAACACCCCTCCGCGCTCTTTTCCGATTTTTTTCTAAACCGTGGAATATTTCG GATATCCTTTTGTTGTTTCCGGGTGTACAATATGGACTTCCTCTTTTCTGGCAACCAAACCCATACATCGGGATT CCTATAATACC7TCGTTGGTCTCCCTAACATGTAGGTGGCGGAGGGGAGATATACATAGAACAG? TACCAGACA AGACATAATGGGCTAAACAAGACTACACCAATTACACTGCCTCAT7G? TGGTGGTACATAACGAACTAATACTGT AGCCCTAGACTTGATAGCCATCATCATATCGAAGTTTCACTACCCTTTTTCCATTTGCCATCTATTGAAG7AATA A7AGGCGCATGCAACTTCTTTTCTTTTTTTTTCTTTTCTCTCTCCCCCGTTG7TGTCTCACCATATCCGCAATGA CAAAAAAAATGATGGAAGACACTAAAGGAAAAAATTAACGACAAAGACAGCACCAACAGATGTCGTTGTTCCAGA GCTGATGAGGGGTATCTTCGAACACACGAAACTTTTTCCTTCCTTCATTCACGCACACTACTCTCTAATGAGCAA CGGTATACGGCCTTCCTTCCAGTTACTTGAATTTGAAATAAAAAAAGTTTGCCGCTTTGCTATCA? GTATAAATA GACCTGCAATTATTAATCTTTTGTTTCCTCGTCATTGTTCTCGTTCCCTTTCTTCCTTGTTTCTTTTTCTGCACA ATA7TTCAAGCTATACCAAGCATACAATCAACTCCAAGCTTGAAGCAAGCCTCC7GAAAGA7GAAGCTACTGTCT TCTATCGAACAAGCATGCGATATTTGCCGACTTAAAAAGCTCAAGTGCTCCAAAGAAAAACCGAAGTGCGCCAAG TGTCTGAAGAACAACTGGGAGTGTC GCTACTCTCCCAAAACCAAAAGGTCTCCGCTGAC7AGGGCACATCTGACA ari? GTGGAATCAAGGCTAGAAAGACTGGAACAGCTATTTCTACTGATTTTTCCTCGAGAAGACCTTGACATGATT TTGAAAA7GGATTCT7TACAGGATATAAAAGCATTGTTAACAGG? TTATTTGTACAAGATAATGTGAATAAAGAT GCCGTCACAGATAGATTGGCTTCAGTGGAGACTGATATGCCTCTAACATTGAGACAGCATAGAATAAGTGCGACA 7CATCATCGGAAGAGAGTAGTAACAAAGGTCAAAGACAGTTGACTGTATCGTCGAGGTCGACCCCGGGTGCTAGC ^ AGGCCTTGTGGCCAGCCATGGCAACTAGTGCGGCCGCTAAGTAAG7AAGACGTCGAGCTC7AAGTAAGTAACGG CCGCCACCGCGGTGGAGCTTTGGACTTCTTCGCCAGAGGTTTGG7CAAGTCTCCAA7CAAGGTTGTCGGCTTG7C 7ACCTTGCCAGA.AATTTACGAAAAGATGGAAAAGGGTCAAATCGTTGGT? GATACG7TGTTGACACTTCTAAA7A AGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGT7ATAAAAAAAATAAGTGTATACAAATTTTAAAG 7GACTCTTAGGTTTTAAAACGAAAATTCTTGTTCTTGAGTAACTCTTTCCTGTAGGTCAGG7TGCTTTCTCAGGT ATAGCATGAGGTCGCTCTTATTGACCACACCTCTACCGGCATGCCGAGCAAATGCCTGCAAATCGCTCCCCA7TT CACCCAATTGTAGATATGCTAACTCCAGCAATGAGTTGATGAATCTCGGTGTGTATTTTATGTCCTC? G? GGACA ATACCTGTTGTAA7CGTTCTTCCACACGGATCCCAATTCGCCCTATAGTGAGTCGTAT7ACAATTCAC7GGCCGT CGTTTT? CAACGTCGTGACTGGGAAAACCCTGGCG TTACCCAACTTAATCGCCTTCCAGCACATCCCCCTT7CGC CAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCC ?? C? GTTGCGCAGCCTGAATGGCGAATGGAC GCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGG7TACGCGCAGCGTGACCGCTACACTTGCCAGCGCC CTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACG '.' TCGCCGGCTTTCOCCGTCAAGCTCTAAAT CGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAA ".CTTGATTAGGGTGATGGT 7CACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTT7GACGTTGGAGTCC? CGTTCTTTAATAGTGGA CTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATA ^ GGGATTTTGCCGATT TCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAA7TTTAACAAAATATTAACGTTTAC? ATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAGGCAAGTGCACAAACAATACT TAAATAAATACTACTCAGTAATAACCTATTTCTTAGCATTTTTGACGAAATTTGCTATTTTGTTAGAGTCTTTTA CACCATTTGTCTCCACACCTCCGCTTACATCAACACCAATAACGCCATTTAATCTAAGCGCATCACCAACATTTT CTGGCGTCAGTCCACCAGCTAACATAAAATGTAAGCTTTCGGGGCTCTCTTGCCTTCCAACCCAGTCAGAAATCG AGTTCCAATCCAAAAGTTCACCTGTCCCACCTGCTTCTGAATCAAACAAGGGAATAAACGAATGAGGTTTCTGTG AAGCTGCACTGAGTAGTATGTTGCAGTCTTTTGGAAATACGAGTCTTTTAATAACTGGCAAACCGAGGAACTC7T GG7ATTCTTGCCACGACTCATCTCCATGCAGTTGGACGATATCAATGCCGTAATCAT7GACCAGAGCCAAAACAT CCTCCTTAGGTTGATTACGAAACACGCCAACCAAGTATTTCGGAGTGCCTGAACTATTTTTATATGCTTTTACAA GACTTGAAATTTTCCTTGCAATAACCGGGTCAATTGTTCTCTTTCTATTGGGCAC? CATATAATACCCAGCAACT CAGCATCGGAATCTAGAGCACATTCTGCGGCCTCTGTGCTCTGCAAGCCGCAAACTTTCACCAATGGACCAGAAC TACCTGTGAAA7TAATAACAGACATACTCCAAGCTGCCTTTGTG7GCTTAATCACGTAT? CTCACGTGCTCAATA GTC? CCAATGCCCTCCCTCTTGGCCCTCTCCTTTTCTTTTTTCGACCGAATTAATTCTTAATCGGCAAAAAAAGA AAAGC7CCGGA7CAAGATTGTACGTAAGGTGACAAGCTATTTTTCAATAAAGAATATCTTCCACTACTGCCA7CT GGCGTCATAACTGCAAAGTACACATATATTACGATGCTGTCTATTAAATGCTTCCTA7ATTATATATATAGTAAT GTCGTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACAC CCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCT GCATGTGTCA3AGGTTTTCACCGTCATCACCGAAACGCGCGA Nucleotide sequence of the recombinant plasmid pDBTRP-MET25-SPC100-Gal4-VP16 (SEQ ID NO: 12): ACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGGACGGATCGCT TGCCTGTAACTTACACGCGCCTCGTATCTTTTAATGATGGAATAATTTGGGAATTTACTCTGTGTTTATTTATTT TTATGTTTTGTATTTGGATTTTAGAAAGTAAATAAAGAAGGTAGAAGAGTTACGGAATGAAGAAA ?? AAAATAAA CAAAGGTTTAAAAAATTTCAACAAAAAGCGTACTTTACATATATATTTATTAGACAAGAAAAGCAGATTAAATAG ATATACATTCGATTAACGATAAGTAAAATGTAAAATCACAGGAT7TTCGTGTGTGGTCT7CTACACAGACAAGAT GAAACAATTCGGCATTAATACCTGAGAGCAGGAAGAGCAAGATAAAAGGTAGTATTTGTTGGCGATCCCCCTAGA GTCTTTTACATCTTCGGAAAACAAAAACTATTTTTTCTTTAATTTCTT7TTTTACTTTCTATTTTTAATTTATAT ATTTATATTAAAAAATTTAAATTATAATTATTTTTATAGCACGTGATGAAAAGG? CCC? GG7GGCACTTTTCGGG GAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCA? ATATGTATCCGCTCATGAGACAATAAC CCTGATAAATGCTTCAATAATCTGCAGCTCTGGCCCGTGTCTCAAAATCTCTG? TGTT? C? TTGC? C ?? GATAAA AATATATCA7CATGAACAATAAAACTGTCTGCTTACATAAACAGTAA7ACAAGGGGTGTTATGAGCCATATTCAA CGGGAAACGTCTTGCTGGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGC GATAATGTCGGGCAATCAGGTGCGACAATCTTTCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAA CATGGCAAAGGT AGCGTTGCCAATGATGTTACÁGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCT CTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCGCGGGAAAACA GCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGG TTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGA ATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTG7TGA ^ CAAG7C7GGAAA GAAATGCATACGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATT TTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCC ATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAaAATATGGTATTGATA? T CCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGT7TTTC7AATCAGAATTGGTTAATTGGTTGT? A CACTGGCAGAGCAT7ACGCTGACTTGACGGGACGGCGCATGACCAAAATCCCTTAACGTGAGTTTTCGT7CCACT GAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGC AAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAAC7CTTTTTCCGAAGGT ?? CTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACT CTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCG TAAGTCGTG7C TTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACAC AGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTAC GCGTGAGCATTGAGAAAGCGCCACGCTTC CCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCCCACGAGGGAGCTTCCAG GGGGGAACGCCTGG7ATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGA7TTTTGTGATGCT CGTC GGGGGGCCGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTT T7G:? TCACATGTTCTTTCCTGCGTTATCCCCTGATTCTG7GGATAACCGTATTACCGCCTTTGAGTGAGCTGATA CCGCTCGCCCCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAAC CGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGC CG C GGTTTCCCG CTGGAAAGCGGGCAGTG AGCGCAACGCAATTAATGTGAGTTACCTCACTCATTAGGCACCCCAGGCTTTACACT7TAT CTTCCGGCTCCT???? TGT7GTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGGAA T7 ?? CCCTC? CTAAAGGGAACAAAAGCTGGTACCGATCCCGAGCTTTGCAAATTAAAGCCTTCGAGCGTCCCAAA ACCTTCTCAAGCAAGGTTTTCAGTATAATGTTACATGCGTACACGCGTCTGTACAGAAAAAAAAGAAAAATTTGA AATATAAATAACGTTCTTAATACTAACATAACTATAAAAAAATAAATAGGGACCTAGACTTCAGGTTGTCTAACT CCT7CCTTTTCGGTTAGAGCGGATGTGGGGGGAGGGCGTGAATGTAAGCGTGACATAACTAATTACATGATATCG ACAAAGGAAAAGGGGCCTGT7TACTCACAGGCTTTTTTCAAGTAGGTAATTAAGTCG7TTCTGTCTTTTTCCTTC TTCAACCCACCAAAGGCCATCTTGGTACTTT7TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCATAGAAATAATACAGAAGTAGATGTTG ?? TT? GATTAAACTGAAGATATATAATTTATTGGAAAA7ACATACAGCTTTTTGTTGA7GCGCTTAAGCG? TC ?? , RTCAACA? C? CCACCAGCAGCTCTGATTTT7TCTTCAGCCAACTTGGAGACGAATCTAGCTTTGACGATAAC7GG AACATTTGGAATTCTACCCTTACCCA? GATCTTACCGTAACCGGCTGCCAAAGTGTCAATAACTGGAGCAGTTTC C7TAGAAGCAGATTTCAAGTATTGGTCTCTCTTGTCTTCTCGGATCAATGTCCACAAT7TGT CAAGTTCA? G? C T3GCTTCCAGAAATGAGCTTGTTGCTTGTGGAAGTATCTCATACCAACCTTACCGAAATAACCTGGATGGTATTT A7CCATG7TAATTCTGTGGTGATGTTG? CC? CCGGCCATACCTCTACCACCGGGGTGCTTTCTGTGCTTACCGAT ACGACCTTTACCGGCTGAGACGTGACCTCTGTGCTTTCTAGTCTTAGTGAATCTGGAAGGCATTCTTGATTAGTT GGATGATTGTTCTGGGATTTAATGCAAAAATCACTTAAGAAGGAAAATCAACGGAGAAAGCAAACGCCATCTTAA ATATACGGGATACAGATGAAAGGGTTTGAACCTATCTGGAAAATAGCATTAAACAAGCGAAAAACTGCGAGGAAA AT7GTTTGCGTCTCTGCGGGCTATTCACGCGCCAGAGGAAAATAGGAAAAATAACAGGGC? T7AGAAAAATAATT TTGATTTTGGTAATGTGTGGGTCCTGGTGTACAGATGTTACATTGGTTACAGTACTCTTGTTTTTGCTGTGTTTT TCGATGAATCTCCAAAATGGTTGTTAGCACATGGAAGAGTCACCGATGCTAAGTT? TCTCTATGTAAGCTACGTG GCGTGACTTTTGATGAAGCCGCACAAGAGATACAGGATTGGCAACTGCAAATAGAATCTCGGGATCCCCCCTCGA CGGATGCAAGGGTTCGAATCCCTTAGCTCTCATTATTTTTTGCTTTTTCTCTTGAG.GTSGTCACATGA7CGCAA AATGGCAAATGGCACGTGAAGCTGTCGATATTGGGGAACTGTGGTGGTTGGCAAATGACTAATTAAGTTAGTCAA GGCGCCATCCTCATGAAAACTGTGTAACATAATAACCGAAGTGTCCAAAAGGTGGCACCTTGTCCAATTGAACAC GCTCGATGAAAAAAATAAGATATATATAAGGTTAAGTAAAGCGTCTGTTAGAAAGGAAGTTTTTCCT7TTTCTTG CTCTCTTGTCTTTTCATCTACTATTTCCTTCGTGTAATACAGGGTCGTCAGATACATAGATAC ?? ?? TTCTATTACC CCCATCCATACATCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGATATCAAGCT7CACAGCTAGCGCACT CGGTGCCCCGCGCAGGGTCGCGATGCTGCCCGGTTTGGCACTGTTCCTGCTGGCCGCCTGGACGGCTCGGGCGCT GGATGCAGAATTCCGACATGACTCAGGATATGAAGTTCATCATCAAAAATTGGTGTTCTTTGCAG GATGTGGG TTCAAACAAAGG TGCAATCATTGGACTCATGGTGGGCGGTGTTGTCATAGCGACAGTGATCGTCATCACCTTGGT -ATGCTGAAGAAGAAACAGTACACATCCATTCATCATGGTGTGGTGGAGGTTGACGCCGCTGTCACCCCAGAGGA GCGCCACCTGTCCAAGATGCAGCAGAACGGCTACGAAAATCCAACCTACAAGTTCT7TGAGCAGATGCAGAACGC GCGGGGTACCCCGGCGATGAAGCTACTGTCTTCTATCGAACAAGCATGCGATATTTGCCGACTTAAAAAGCTCAA GTGCTCCAAAGAAAAACCGAAGTGCGCCAAGTGTCTGAAGAACAACTGGGAGTGTCGCTACTCTCCCAAAACCAA AAGGTCTCCGCTGACTAGGGCACATCTGACAGAAGTGGAATCAAGGCTAGAAAGACTGGAACAGCTATTTCTACT GATTTTTCCTCGAGAAGACCTTGACATGATTTTGAAAATGGATTCTTTACAGGATATAAAAGCATTGTTAACAGG ATTATTTC7ACAAGATAATGTGAATAAAGATGCCGTCAC? GATAGATTGGCTTCAGTGG? GACTGATATGCCTCT AACATTGAGACAGCATAGAATAAGTGCGACATCATCATCGGAAGAGAGTAGTAACAAAGGTCAAAGACAGTTGAC TGTATCGCCGGAATTCCCGGGGATCTGGGCCCCCCCGACCGATGTCAGCCTGGGGGACCACCTCCACTTAGACCG CGAGGACGTGGCGATGGCGCATGCCGACGCGCTAGACGATTTCGATCTGGACATGTTGGGGGACGGGGATTCCCC GGGGCCGGGATTTACCCCCCACGACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTTCGAGTTTGAGCAGAT GTTTACCGATGCCCTTGGAATTGACGAGTACGGTGGGTAGGGATCCACTAGTCCAG7GTGGTGGAATTCTGCAGA lATCCAGCACAGTGGCGGCCGctc gaCCCCGGGTGCTAGCAAGGCCTTGTGGCCACCCATCGCAACTAGTGCGGC CGCTAAGTAAG7AAGACGTCGAGCTCTAAGTAAGTAACGGCCGCCACCGCGGTGGAGCTTTGG? CTTCTTCGCCA GAGGTTTGGTCAAGTCTCCAATCAAGGTTGTCGGCTTGTCTACCTTGCCAGAAATTTACGA A? G? TGGA ?? s ?? AGG GTCAAATCGTTGGTAGATACGTTGTTGACACTTCTAAATAAGCGAATTTCTTATGATTTATGATTTTTATTA7TA AATAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACG7 TTCTTGTTCT rGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGCATGAGGTCGCTCTTATTCACCACACCTCTA CCGGCATGCCGAGCAAATGCCTGCAAATCGCTCCCCATTTCACCCAATTGTAGATATGCTA? CTCCAGCAATGAG T7GATGAATCTCGGTGTGTATTTTATGTCCTCAGAGGACAATACCTGTTGTAATCGTTCTTCC? C? CGG? TCCCA ATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCG TTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATC GCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGT GGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTT TCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTT ACGGCACCTCGACCCCAAAAAACTTGATTAGGGTG ATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTT TCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACAC7CAACCCTAT CTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACA AAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTG 7GCGGTATTTCACACCGCAGGCAAGTGCACAAACAATACTTAAATAAATACTACTCAGTAATAACCTATTTCTTA GCATTTTTGACGAAATTTGCTATTTTGTTAGAGTCTTTTACACCATTTGTCTCCACACCTCCGCTTACATCAACA CCAATAACGCCATTTAATCTAAGCGCATCACCAACATTTTCTGGCGTCAGTCCACCAGCTAACATAAAATGTAAG CTT7CGGGGCTCTCTTGCCTTCCAACCCAGTCAGAAATCGAGTTCCAATCCAAAAGTTCACCTGTCCCACCTGCT TCTGAATCAAACAAGGGAATAAACGAATGAGGTTTCTGTGAAGCTGCACTGAGTAGTATGTTGCAGTCTTTTGGA AATACGAGTCTT7TAATAACTGGCAAACCGAGGAACTCTTGGTATTCTTGCCACGACTCATCTCCATGCAGTTGG ACGATATCAATGCCGTAATCATTGACCAGAGCCAAAACATCCTCCTTAGGTTGATTACGAAACACGCCAACCAAG TATT7CGGAGTGCC7GAACTATTTTTATATGCTTTTACAAGACTTGAAATTTTCCTTGCAATAACCGGGTCAATT GTTCTCTTTCTATTGGGCACACATATAATACCCAGCAAGTCAGCATCGGAATCTAGAGCACATTCTGCGGCCTCT GTGCTCTGCAAGCCGCAAACTTTCACCAATGGACCAGAACTACCTGT GAAA7TAATAACAGACATACTCCAAGCT GCCTTTGTGTGCTTAATCACGTATACTCACGTGCTCAATAGTCACCAATGCCCTCCCTCTTGGCCCTCTCCTTTT CTTTTTTCGACCGAATTAATTCTTAATCGGCAAAAAAAGAAAAGCTCCGGATCAAGATTGTACGTAAGGTGACAA GCTATTTTTCAATAAAGAATATCTTCCACTACTGCCATCTGGCGTCATAACTGCAAAGTACACATATATTACGAT GCTGTCTATTAAATGCTTCCTATATTATATATATAGTAATGTCGTTTATGGTGCAC7CTCAGTACAATCTGCTCT GATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGC7GACGCGCCCTGACGGGCTTGTCTGCTCCCG GCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAA CGCGCGA Amino acid sequence of a fused protein SP-C100-Gal4-VP16 (SEQ ID NO: 13): MLPGLALFLL AAWTARALDA EFRHDSGYEV HHQKLVFFAE DVGSNKGAI I GLMVGGWIA TVIVITLVML KKKQYTSIHH GVVEVDAAVT PEERHLSKMQ QNGYENPTYK FFEQMQNARG TPAMKLLSSI EQACDICRLK KLKCSKEKPK CAKCLKNNWE CRYSPKTKRS PLTRAHLTEV ESRLERLEQL FLLIFPREDL DMILKMDSLQ DIKALLTGLF VQDNVNKDAV TDRLASVETD MPLTLRQHRI SATSSSEESS NKGQRQLTVS PEFPGIWAPP TDVSLGDELH LDGEDVAMAH ADALDDFDLD MLGDGDSPGP GFTPHDSAPY GALDMADFEF EQMFTDALGI DEY GG References: Estus et al. (1992), Science, 255, 726. Haass et al. (1992) Nature, 359, 322. Hilbich et al. (1991) J. Mol. Biol., 218, 149 Kang et al. (1987) Nature, 325, 733 Maruyama et al. (1994) Biochem. Biophys Res Commun, 202, 1517 Rumble et al. (1989), N. Engl. J. Med., 320, 1446 Sadowski et al. (1988) Nature, 335, 563 Scheuner et al. (1996), Nature Medicine, 2, 864 Simons et al. Neurosci (1996) 1; 16 (3): 899-908 Suzuki et al. Science 1994 May 27; 264 (5163): 1336-40 Yankner et al. (1990) Proc Nati Acad Sci USA.87, 9020

Claims (23)

1. CLAIMS 1. A process for the detection of? -secretase activity, where I. a transgene is used which encodes a fused protein and contains the following constituents: a) a first nucleotide sequence that encodes a protein containing the amino acid sequence GAIIGLMVGGVVIATVIVITLVML (SEQ ID NO: 1), b) At the 5 'end of the first nucleotide sequence, a second nucleotide sequence coding for a signal peptide, c) a promoter y, d) if appropriate, additional nucleotide sequences that encode and / or do not code. II. this transgene is incorporated within a cell and the fused protein is expressed; III. The fused protein is split with the amino acid sequence SEC. FROM IDENT. 1 for the β-secretase present in the cell, with which a first partial protein is formed, which contains the amino acid sequence GAIIGLMVGGVV (SEQ ID NO: 2), and a second partial protein, which contains the sequence of amino acids VIVITLVML (SEQ ID NO: 3), and IV. The partial protein and / or the second partial protein are detected. 2. A process for the detection of? -secretase activity, where I. a transgene is used which encodes a fused protein and contains the following constituents: a) a first nucleotide sequence that encodes a protein containing the sequence of amino acids GAIIGLMVGGVVIATVIVITLVML (SEQ ID NO: 1), b) At the 5 'end of the first nucleotide sequence, a second nucleotide sequence encoding a signal peptide, c) a promoter, and d) if appropriate , additional nucleotide sequences that encode and / or do not code. II. this transgene is incorporated within a cell and the fused protein is expressed; II. The fused protein is split with the amino acid sequence SEC. FROM IDENT. DO NOT. 1 for the β-secretase present in the cell, with which a first partial protein is formed, which contains the amino acid sequence GAIIGLMVGGVV (SEQ ID NO: 2), and a second partial protein, which contains the sequence of amino acids VIVITLVML (SEQ ID NO: 3) and IV. the amount of the second partial protein is determined and the activity of the β-secretase is determined from the amount of the second partial protein formed. 3. The process as claimed in one of claims 1 and 2, wherein the first nucleotide sequence encodes a precursor amyloid protein (APP) or a portion thereof. 4. The process as claimed in one or more of claims 1 to 3, wherein the first nucleotide sequence encodes a protein having the amino acid sequence SEQ. FROM IDENT. DO NOT. 4. The process as claimed in one or more of claims 1 to 4, wherein the second nucleotide sequence codes for the APP (SP) signal peptide. The process as claimed in one or more of claims 1 to 5, wherein the signal peptide has the amino acid sequence SEC. FROM IDENT. DO NOT. 5. The process as claimed in one or more of claims 1 to 6, wherein the promoter is a promoter for expression in mammalian cells, in C. elegans, in yeast or in Drosophila. The process as claimed in one or more of claims 1 to 7, wherein the promoter is the CMV promoter, HSV TK, RSV, SV40, LTR, uncll9, unc54, hspl6-2, G0A1, sel-12, ADH1, Gall, MET3, Met 25, MT, Ac5 or Ds47. 9. The process as claimed in one or more of claims 1 to 8, wherein the cell is a eukaryotic cell. 10. The process as claimed in one or more of claims 1 to 9, wherein the cell is a human cell. The process as claimed in one or more of claims 1 to 9, wherein the cell is a non-human cell. 12. The process as claimed in claim 11, wherein the cell is a HeLa cell, 293, H4, SH-SY5Y, H9, Cos, CHO, N2A, SL-2 or Saccharomyces cerevisiae. 13. The process as claimed in claim 11, wherein the cell is a C. elegans cell. The process as claimed in claim 12, wherein the cell is a constituent of a transgenic C. elegans. 15. The process as claimed in claim 11, wherein the cell is a yeast cell. The process as claimed in one or more of claims 1 to 15, wherein the fused protein has or contains the amino acid sequence SEQ. FROM IDENT. DO NOT. 6. The process as claimed in one or more of claims 1 to 16, wherein the additionally encoding nucleotide sequence is located at the 3 'end of the first nucleotide sequence. The process as claimed in one or more of claims 1 to 17, wherein the nucleotide sequence it encodes additionally encodes a protein that is expressed as a protein fused to the first partial protein and the second partial protein and can used for the detection of the second partial protein. The process as claimed in one or more of claims 1 to 18, wherein the nucleotide sequence encodes additionally for a protein that contains a DNA binding domain and a transcription activating domain. The process as claimed in one or more of claims 1 to 19, wherein the nucleotide sequence it encodes additionally encodes a protein consisting of a domain that binds Gal4 and the domain that activates transcription of VP16 (Gal4- VP16). The process as claimed in one or more of claims 1 to 20, wherein the cell is co-transfected with a reporter plasmid, wherein the reporter plasmid contains a reporter gene under the control of an adjustable promoter. 22. The process as claimed in claim 21, wherein the regulatable promoter is activatable by the domain that activates transcription. 23. The process as claimed in claim 22, wherein the reporter plasmid encodes the reporter gene for EGFP (Enhanced Green Fluorescent Protein) and the regulatable promoter contains sites that bind Gal4 and a minimal HIV promoter.
2. 2 . The process as claimed in one or more of claims 1 to 23, wherein the transgene has the nucleotide sequence SEC. FROM IDENT. DO NOT. 8. The process as claimed in one or more of claims 1 to 24, wherein the transgene is present in a vector. 26. The process as claimed in one or more of claims 1 to 25, wherein the recombinant vector has the nucleotide sequence SEQ. FROM IDENT. DO NOT. 9. The process as claimed in one or more of claims 1 to 26, wherein endogenous β-secretase activity in the cell is not detectable. The process as claimed in one or more of claims 1 to 27, wherein the cell is co-transfected using a cDNA library. 29. The process as claimed in claim 28, wherein the cDNA prepared from a human or non-human tissue or human or non-human cells is present in the cDNA library. The process as claimed in one or more of claims 27 to 29, for the identification of a cDNA encoding a? -secretase, wherein a) a cell is identified in which the activity of a? -secretase it is detectable and b) the cDNA encoding the β-secretase is isolated from this cell. 31. A transgene, comprising a) a first nucleotide sequence encoding a protein containing the amino acid sequence GAIIGLMVGGVVIATVIVITLVML (SEQ ID NO: 1), b) at the 5 'end of the first nucleotide sequence , a second nucleotide sequence encoding a signal peptide, c) a promoter and d) at least one additional nucleotide sequence at the 3 'end of the first nucleotide sequence, which codes for a domain that links DNA and for a domain that activates transcription. 32. The transgene as claimed in claim 31, wherein the first nucleotide sequence codes for APP or a portion of APP. 33. A transgene as claimed in one or more of claims 31 and 32, wherein the transgene has the nucleotide sequence SEQ. FROM IDENT. DO NOT. 8. 34. A vector comprising a transgene as claimed in one or more of claims 31 to 33. 35. The vector as claimed in claim 34, wherein the vector has the nucleotide sequence SEQ. FROM IDENT. DO NOT. 12. 36. A process for the production of a transgenic cell, characterized in that a cell is transfected with a vector as claimed in one or more of claims 34 and 35. 37. A process for the production of a transgenic C. elegans , wherein a transgene as claimed in one or more of claims 31 to 33 is microinjected into the gonads of a C. elegans. 38. A cell comprising a transgene as claimed in one or more of claims 31 to 33. 39. A transgenic C. elegans comprising a transgene as claimed in one or more of claims 31 to 33. 40. A yeast cell comprising a transgene as claimed in one or more of claims 31 to 33. 41. A cell comprising a) a transgene as claimed in one or more of claims 31 to 33; b) a cDNA library, and c) a reporter plasmid 42. The use of a cell as claimed in claim 41, for the identification of a cDNA encoding a gamma secretase. 43. A process for the identification of a? -secretase cDNA, wherein I) a cell as claimed in claim 41 is produced, and II) is determined if a second partial protein is formed. 44. The use of a cell as claimed in claim 41, in a process for the identification of inhibitors of the activity of a β-secretase. 45. A process for the identification of substances that inhibit the activity of a? -secretase, wherein the process comprises the following stages of the process: I. Production of a transgenic non-human organism or a transgenic cell, where the organism does not The transgenic human or the transgenic cell contains a transgene having the following constituents: a) a first nucleotide sequence encoding a protein containing the amino acid sequence GAIIGLMVGGVVATVIVITLVML (SEQ ID NO: 1), b) at the extreme 5 'of the first nucleotide sequence, a second nucleotide sequence coding for a signal peptide, c) a promoter and, d) if appropriate, additional nucleotide sequences which do not encode and / or encode; contains a reporter plasmid carrying a protein binding site, a minimal promoter and a reporter gene, and, if appropriate, a cDNA encoding a β-secretase wherein the transgenic non-human organism or the transgenic cell expresses the transgene and, if appropriate, the? -secretase encoded by the cDNA; II. the transgenic non-human organism or the transgenic cell is incubated with a substance that is to be investigated and III. the amount of the second partial protein is determined. 46. A process for identifying substances that inhibit the activity of a? -secretase, where I. A transgene containing the following constituents is used: a) a first nucleotide sequence encoding a protein containing the amino acid sequence GAIIGLMVGGVVIATVIVITLVML (SEQ ID NO: 1), b) at the 5 'end of the first nucleotide sequence, a second nucleotide sequence encoding a signal peptide and c) a promoter and, d) if appropriate, additional nucleotide sequences encoding and / or not coding; II. this transgene and a reporter plasmid and, if appropriate, a cDNA encoding a? -secretase are incorporated into a cell and the fused protein encoded by the transgene and, if appropriate, the? -secretase encoded by the cDNA is they express in the presence of a substance that is going to be investigated. III. the fused protein is either a) unfolded, whereby a first partial protein containing the amino acid sequence GAIIGLMVGGVV (SEQ ID NO: 2) and a second partial protein containing the amino acid sequence VIVITLVML (SEC) is formed. DE IDENT NO 3), or b) unfolded, whereby a detectable amount of the first and / or second partial protein is not formed within the amino acid sequence SEC. FROM IDENT. DO NOT. 1 for the? -secretase present in the cell, IV. the amount of the second partial protein is determined. 47. A process for the identification of substances that inhibit the activity of a β-secretase, wherein a transgene encodes a fused protein containing a signal peptide and SEC. FROM IDENT. DO NOT. 1 is expressed in the presence of a substance to be investigated and the effect of the substance to be investigated is determined on the amount of the second partial protein formed, containing the second partial protein, the amino acid sequence VIVITLVML (SEC IDENTITY NO 3). 48. A process for the production of a drug, comprising a process as claimed in one or more of claims 45 to 47 and finally to the identified inhibitor mixture with a pharmaceutically tolerable carrier. 49. A process as claimed in one or more of claims 45 to 47, further comprising the formulation of the identified inhibitor in a pharmaceutically tolerable form. 50. A test kit for the qualitative and / or quantitative detection of a β-secretase activity comprising a fused protein containing the amino acid sequence SEC. FROM IDENT. NO 1