HUT76973A - Method for detection of malfolded protein - Google Patents

Description

Procedure for Detecting False Folded Protein

The present invention relates to a method for detecting misfolded proteins, to a method for measuring the amount of misfolded protein produced, and to a method for detecting compounds which affect folding.

When incorrectly folded proteins accumulate in a cell, transcription of genes encoding certain proteins is induced in the nucleus. This misfolded protein response was first studied in animal cells, where the expression of BIP / GRP18, GRP94, PDI / ERp59 and ERp72 is induced by various treatments that increase the amount of misfolded protein. The response to accumulation of misfolded proteins is regulated at the transcriptional level by certain enhancer elements.

A similar response has been observed to the accumulation of misfolded proteins in Saccharomyces cerevisiae, where expression of a similar gene pool is induced, including Bip (encoded by KAR2), PDI, Euglp and peptidyl-prolyl cistrans isomerase. Using a KAR2 promoter deletion analysis, a 22 bp misfolded protein response element (UPR) was identified. This UPR is necessary and sufficient in response to the accumulation of misfolded protein

-2 · ·· · · for active transcription (Shamu et al., Trends in Cell Bioi. (1994), 4, 56-60).

The biological function of a protein is controlled by specifically upregulated three-dimensional structures. Misfolded or aggregated proteins fail to obtain the required conformation required for the activity of a protein. Generally, the accumulation of misfolded proteins in the cell is directly or indirectly associated with serious diseases such as Alzheimer's disease (AD) and human prion-related diseases such as Creutzfeld-Jacob disease and Gertsmann-Strasse-Scheinker disease. Another example of the effect of a misfolded protein is the p53 suppressor protein, a negative regulator of cell growth (Donehower & Bradley, Biochim. Biophys. Acta (1993) 1155, 81-205). Most human tumors express mutant p53 proteins. Therefore, mutant p53 proteins are presumed to be flawed or at least partially denatured.

For example, Alzheimer's disease is associated pathologically with cerebrovascular amyloid deposits. The major protein found in these deposits is a peptide of 39 to 42 amino acids, called the β-amyloid peptide, and is predominantly in the cross β conformation. CDNA analysis revealed that the β-amyloid peptide was derived from an amilord precursor protein (APP) by rn fusing proteolysis. APP is produced in many cell types and normally does not aggregate. In contrast, β-amyloid synthesis molecules have been shown to aggregate,

-3 (Barrow et al., J. Mol. Biol. (1992), 225, 1075-1093). It is believed that in the cell, the proteins that initially misfold are necessarily aggregated, but the proteins that aggregate are not necessarily misfolded.

With this knowledge, it would be extremely valuable to have a test that would allow a quick and easy examination of the effect of compounds on misfolding of proteins, for example, their ability to minimize the production of a certain misfolded protein, or counteract the effects that cause a protein to be folded incorrectly.

Surprisingly, it has been found that the effect of compounds on misfolding of proteins can be demonstrated through an easily measurable reporter gene product when DNA encoding the misfolded protein is operably linked to a signal sequence that triggers transport of this protein to the endoplasmic reticulum (ER). ) and if one or more UPR elements are operably connected to the reporter element. The method of the present invention also provides an easy method for determining whether an abnormal aggregation of a protein is associated with a defective folding or not. For example, in the case of the β-amyloid peptide, it has surprisingly been found that aggregation, which occurs both in vivo and in vitro, is associated with malfunctioning and, using the method of the invention,

-4vivo is traceable. Furthermore, the degree of aggregation of the various β-amyloid peptides (truncated, inverted, or derived from different organisms), based on in vitro estimation, is closely correlated with the degree of defective folding measured by the method of the invention. Therefore, it is also possible to observe the effect of mutations on the degree of defective folding and their associated effects.

The invention relates to a host transformed with at least one first and second expression cassettes, wherein the first expression cassette comprises one or more misfolded protein response elements (UPR) operably linked to a reporter element;

the second expression cassette comprises a promoter operably linked to a signal sequence, a DNA encoding a protein whose folding is to be observed, and a terminator; and wherein said first and second expression cassettes are not naturally occurring in the host, and wherein the protein to be monitored in the second expression cassette is selected from the group consisting of a prion, p53, β-amyloid peptide, and functional derivatives thereof.

hosts

A suitable host organism of the invention may be any host capable of responding to a flawed protein response, such as a host that secretes proteins through the ER. Suitable host organisms include, for example, plant, insect, mammalian, fungi, or

-5 animal cells. Fungal cells are preferred, more preferably a yeast and most preferred is Saccharomyces cerevísiae.

Suitable yeast strains of the invention include strains of S. cerevisiae containing endogenous two micron plasmids and strains treated with said endogenous two micron plasmid (see EP-A-340170).

The host organism can be transformed with the required first and second expression cassettes using conventional techniques in genetic engineering, such as those described in Sammbrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, 1989.

Expression cassettes

The misfolded protein response element (UPR), as used in the first expression cassette, is a fragment of DNA involved in initiating transcription upon accumulation of misfolded protein. For example, UPR elements may be isolated from the initiation region of a protein that is originally involved in the response to misfolded protein accumulation, such as BiP, FKB2, BiP / GRPU, GRP94, PDI / ERp59 and ERp72 (Shamu et al., Trends in Cell Biol. (1994), 4, 56-60).

Preferably, a UPR is isolated from BiP, and more preferably, it comprises a DNA sequence as set forth in SEQ ID NO: 1 or a functional equivalent thereof. Functional

-6 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · .

which do not affect the activity of the response to folded protein accumulation. For a more powerful response to accumulation, the UPR may be present in multiple copies, such as 2 to 5 copies.

According to the invention, in the preferred expression cassette, the reporter element comprises a promoter operably linked to a DNA that is transcribed under the control of that promoter and a terminator.

The promoter in this reporter element can be of almost any origin. For example, it is possible to have a tightly regulated promoter or a promoter that is naturally adjacent to said DNA, such as the β-galactosidase or luciferase promoter or the CYC1, Gall / GAL19, PHO5 or KAR2 promoter. In a preferred embodiment of the invention, the promoter is selected from the CYC1 and KAR2 promoters.

Suitable DNA transcribed under the control of this promoter generally produces effects that are readily measurable during or after transcription or translation. Preferred are transcriptional or translational products which are readily measurable, for example by measuring the amount of protein, mRNA or DNA produced; or those that produce an effect that is easily measurable, such as cell growth, an enzymatic reaction or a color. Therefore, for example, the transcribed DNA encodes a protein that is produced in an amount that is proportional to its degree and that of said promoter, which, for example, is not produced elsewhere in the selected host under the conditions used. For example, suitable proteins include metallothionein, encoded by the yeast CUP1 gene, β-galactosidase or luciferase. Luciferase and β-galactosidase are preferred.

A terminator suitable for this first expression vector generally also contains signals corresponding to transcription termination. This terminator may be a terminator naturally associated with the transcribed DNA or a terminator incorporated from a different source such as PHO5, α-factor or SUC2 terminator.

This first expression cassette may further comprise, for example, a DNA sequence encoding a signal peptide as defined above.

The promoter of the second expression cassette of the present invention can be almost any promoter homologous or heterologous to the host. For example, it is possible to use a promoter that is originally linked to DNA encoding a protein whose folding is desired to be detected; or it may be a promoter commonly used in genetic manipulation of the host. The promoter may be inducible or non-inducible, such as CUP1,

GAPDH, GAPFL, GAL (1/10), PYK, TPI, ADH, PRC1 and the PGK promoter. GAPFL promoter and functional derivatives thereof are preferred.

When an inducible promoter is used, the host is grown under optimal culture conditions and the expression of the protein whose folding is desired can be induced at the desired moment.

The signal sequence is generally selected according to the host used in the assay. A suitable signal sequence can be derived from any gene encoding a polypeptide that is secreted in a conventional manner. These include SUC2, CPY, α-factor, KEX1, PHO5 and the glucoamylase signal sequence.

Proteins whose defective fold inhibition is to be detected are prions, p53, β-amyloid peptide or functional derivatives thereof. In a preferred embodiment of the invention, it is desired to detect a defective fold inhibition of the protein milk by the β-amyloid peptide or functional derivative thereof. The term functional derivative refers to a peptide derived from the original misfolded peptide with minor alterations, such as 1 to 10 amino acid substitutions, additions or deletions, but such that the properties of the folding are comparable or the same.

The terminator used in the second expression cassette may be a terminator naturally associated with the DNA encoding the protein whose folding is to be detected, or another terminator as described above.

The promoter, the DNA sequence encoding the signal peptide, the DNA sequence encoding the polypeptide, and transcription termination signals containing

The DNA sequence * is functionally linked to each other in a manner that preserves their function. The sequence is such that the signal sequence - polypeptide gene complex results in the proper expression of the normal promoter, the signals result in transcription and termination, and the transcription termination in the correct signal sequence of the polyadenylation is linked in the correct reading frame to the is directly linked to the first codon of the polypeptide gene. Preferably, the promoter is a major mRNA start site and is naturally linked to the promoter gene

ATG between signal sequences. The signal sequence has its own ATG to initiate translation. The linkage of these sequences can be accomplished, for example, with synthetic oligonucleotide linkers that carry the recognition sequence of an endonuclease.

The expression cassettes of the invention may be inserted into the host genome or in the form of a stable plasmid, such as a two micron plasmid. When one or both expression cassettes are inserted into a stable plasmid, the expression plasmids may contain, in addition to the polypeptide expression cassettes, a portion of DNA derived from the two-micron plasmid containing the origin of replication or, if a two-micron plasmid-free yeast strain is used. plasmid. In this case, the latter type of plasmids are preferred.

For example, the plasmids of the invention contain a complete, two-stranded, double-stranded DNA, i.e., the double-stranded DNA is simply cleaved by a restriction endonuclease, the linearized DNA being linked to the other components of the vector before re-ringing. The restriction site is chosen to retain the REP1, REP2 and FLP genes of the two-micron DNA and the SRI, STB, IR1 and IR2 sites, as well as the small FLP recognition target sites located near the center of each inverted repeat sequence (IR). , where FLP recombinase acts. Optionally, a restriction site may be selected such that the D-gene of the two-micron DNA remains intact. Suitable restriction sites include, for example, the unique PstI site in the D gene and the unique Hpal and SnaBI sites outside of all said genes and sites. In addition, it is also possible to insert the expression cassette and further components (see below) at different (such as two) restriction sites in the two-micron plasmid, especially the restriction sites mentioned above.

Such a plasmid derivative may contain only two inverted FRT sites or an additional third FRT site. Such a first-mentioned type of plasmid is hereinafter referred to as a symmetric two-micron-like hybrid vector. The latter type of plasmid is hereinafter referred to as a symmetric two-micron disintegration vector, although not a true symmetric plasmid, but a symmetric two-micron microorganism transformed in it.

-11-like hybrid vector (EP-A-501 914).

Preferably, a symmetric two micron-like hybrid vector of the invention does not contain bacterial or viral DNA sequences, i.e. DNA from a bacterial genome, plasmid, or virus. In contrast, a two micron-like disintegration vector of the invention may contain prokaryotic DNA between two directly replicating FRT sites, which is cleaved in the transformed yeast cell, which produces a symmetric two-micron-like hybrid vector from the disintegration vector. These DNA sequences are bacterial sequences described below that may confer essential structural or functional properties to the vector, or may serve only to fill the gap between an inverted repeat FRT site of an asymmetric two-micron-like plasmid or a non-symmetric disintegration vector. two regions to form a symmetric two-micron-like hybrid vector or a symmetric disintegration vector.

In a preferred embodiment, the two regions between the inverted repeat FRT sites of the circular form of the two-micron DNA are of approximately the same length.

Preferably, the expression plasmids of the invention comprise one or more, in particular one or two, selective genetic markers for the host used in the assay, and one marker and an origin of replication (except for the symmetric two-micron-like expression system).

-12bacterial host,

I - · *

I · · · · · · · · (especially hybrid vectors) one

Escherichia coli.

With regard to selective gene markers, any marker gene which permits selection of transformants due to the phenotypic expression of the marker gene can be used. Suitable markers include, for example, those expressing antibiotic resistance or, in the case of auxotrophic host genes, those genes that complement lesions of the host.

Suitable genes are, for example, those conferring resistance to the antibiotic G418, hygromycin or bleomycin, or conferring prototrophy in an auxotrophic yeast mutant, such as the URA3, LEU2, LYS2, HIS3 or TRP1 gene.

Because amplification of expression plasmids can be conveniently performed in a prokaryote, such as E. coli, they preferably include a prokaryotic, e.g., E. coli, genetic marker, and a prokaryotic, e.g., E. coli origin of replication. They can be obtained from suitable prokaryotic plasmids, for example, E. coli plasmids such as pBR322 or a pUC plasmid, such as pUC18 or pUC19, which both contain prokaryotic, e.g., E. coli origin of replication and antibiotics such as a genetic marker conferring resistance to ampicillin.

In addition to the polypeptide expression cassette, the origin (s) of replication, and the genetic marker (s), the expression plasmids of the invention may optionally be <* · ·· ♦ ·

-13 · · · · expression cassettes, such as 1-3 additional polypeptide expression cassettes.

The expression plasmids of the invention are prepared by methods known in the art, for example by replicating the polypeptide expression cassette, DNA fragments containing selective genetic markers for the host used for testing and optionally a bacterial host, replication of the yeast and optionally a bacterial host. and any additional functional fragments or expression cassettes are linked in a predetermined order using conventional chemical or biological in vitro synthesis procedures. Preferably, the plasmids are constructed and prepared using recombinant DNA techniques. For production by recombinant DNA techniques, the appropriate DNA fragments are ligated in vitro by conventional means. The ligation mixture is then transformed into a suitable prokaryotic or eukaryotic host, depending on the control elements used, and the transformant containing the desired vector is selected by conventional methods. The plasmids can be amplified and isolated by conventional means using transformed hosts. The choice of host depends on the regulatory sequences on the vector. Because the expression vectors of the invention preferably contain regulatory sequences operating in prokaryotes such as E. coli, a prokaryotic host such as E. coli

-14colit is preferred for constructing and propagating the vector.

In a further embodiment, the invention relates to an expression cassette comprising a promoter operably linked to a signal sequence, a DNA encoding a β-amyloid peptide or a functional derivative thereof, and a hybrid plasmid comprising said expression cassette. In a preferred form, this plasmid is based on the two micron plasmid of S. cerevisiae.

Methods for constructing these expression cassettes from various functional fragments are well known in the art and are described, for example, in Molecular Cloning: A Laboratory Manual, 2nd edition, 1989 by Sambrook et al.

Transformed strains are cultured using techniques known in the art.

Suitable complex culture media for use in culturing yeast are well known in the art. For example, such culture medium contains tryptone, peptone, meat extract, malt extract, yeast extract, casino acids, corn steep liquor, soybean flour, whey, whey hydrolyzate, etc., and in particular mixtures thereof, and glucose, e.g. , vitamins (such as biotin), unique amino acids, inorganic salts (such as sodium, potassium, magnesium and calcium sulfate, chloride, phosphate, and carbonate),

-15 further with the appropriate salts of trace elements such as iron, zinc, manganese) and the like, taking into account that all of the essential ingredients outlined above are present in the medium. One preferred culture medium is the commercially available YPD medium (yeast extract, peptone, dextrose, see Methods Enzymol. 194, 13), optionally supplemented with inorganic acids and vitamins. Test procedure

In another embodiment, the invention relates to a method of determining the effect of a compound on the appearance of a misfolded protein, wherein a transformed host organism is cultured under the appropriate conditions as defined above, the test compound is introduced and the amount of reporter gene activation is measured.

This effect may be based, for example, on the inhibition of conditions that cause malfunctioning of said protein, on the promotion of correct folding of said protein, or on prevention of aggregation of said protein.

For example, the test may be performed as follows:

culturing the transformed host carrying the two expression cassettes under appropriate conditions;

optionally inducing the expression of the protein whose folding is desired to be observed, such as when the expression of the protein is regulated by an inducible promoter;

• β · · · · ·

-16, if desired, a compound that promotes defective fold inhibition or aggregation is added, for example, if the defective fold of the protein is dependent on additional compounds;

- adding the test compound; and monitoring the transcription or translation of the reporter element or the transcription or translation product or the effects thereof.

In a preferred embodiment of the invention, the method is used to identify compounds which inhibit β-amyloid peptide aggregation.

For example, these compounds may act as follows:

and thereby bind to or modify the protein to inhibit aggregation;

- bind to or modify the protein to inhibit the binding of another compound which initiates or promotes aggregation; obsession

- bind to the compound or modify it to initiate or promote protein aggregation. Compounds, and thus protein, do what

Also included within the scope of the invention are the novel compounds identified using the method of the invention and the use of these compounds in a method of treatment, in particular for inhibiting protein aggregation as defined above. These novel compounds can be used, for example, in the treatment of cancer or Alzheimer's disease.

-17 Brief Description of the Figures

In the following experimental section, various embodiments of the invention will be described with reference to the accompanying drawings:

Figure 1 is a schematic representation of plasmid pCS2-1.

Figure 2 is a schematic representation of pFL38 / CPY.

Examples

The following examples illustrate the invention but should not be construed as a limitation thereof.

All enzymatic reactions, such as restriction enzyme cleavage, ligation, transformation, annealing, and βgalactosidase assay, are standard techniques in genetic engineering and are essentially as described in Sambrook et al., Molecular Cloning: Laboratory Manual (Molecular

Cloning: A Laboratory Manual), 2nd Edition, 1989.

In each PCR reaction Venus was used in the polymerase enzyme (New England Bio-Labs). The primers were synthesized on a DNA synthesizer.

Example 1: Construction of vector pPFY7 for the integration into yeast of lacz expression cassettes regulated by a flawed folding protein response element (s) linked to a core promoter

Plasmid pPFY7 is a vector based on pBluescript (Stratagene®). It encodes the LEU2 gene of Saccharomyces cerevisíae as a yeast selection marker. It also contains the CYClp-lacz fusion gene. CYClp represents the core promoter of the yeast iso-1-cytochrome c (CYC1) gene and lacz encodes the E. coli β-galactosidase enzyme.

Plasmid pPFY7 is obtained as follows.

Plasmid -18A pBluescript SK + (Stratagene®) was digested with SspI and NaIl and a 2832 bp fragment was isolated by agarose / TBE gel electrophoresis. The fragment was isolated and purified with GeneClean® (Bio 101, CA, USA).

The -2190 bp Saccharomyces cerevisiae LEU2 gene was isolated as a SspI-TthI fragment from pRS425 (ATCC 77106). After TRS digestion of pRS425, the sticky end was filled with a large fragment of Klenow DNA polymerase, followed by partial digestion with Sspl. The above fragment was isolated and purified with GeneClean® as described above.

The 2832bp Sspl-Nael fragment and the -2190 bp Sspl-charged fragment were ligated and transformed into E. coli HB101. DNA from individual transformants was analyzed by restriction enzyme analysis. One of the clones with the correct restriction fragments is called pLEU2. Plasmid pLEU2 was completely digested with XhoI and PvuII. An -4716 bp fragment was isolated as described above. An -3500 bp XhoI-PvuII fragment was isolated from plasmid pLG669-Z (Guarente & Ptashne, Proc. Natl. Acad. Sci. USA (1981), 2199-2203) by first digesting with XhoI and then partial. digestion with PvuII. The two fragments were ligated and transformed into HB101. One clone with the correct restriction fragments is called pPFY7.

Example 2: Construction of plasmids containing a lacz expression cassette under the control of a misfolded protein response element (s) linked to a Core promoter

-19Α to 22 bp misfolded protein response (UPR) element (Móri et al., EMBO J (1992), 11, 2583-2593), consisting of double-stranded deoxyoligoryribonucleotide linkers (SEQ ID NO: 1), is chemically synthesized. The fragment contains Xhol sticky ends, one of which remains as an Xhol site after being subcloned into the Xhol digested vector pPFY7. The ligation is performed after the linker tail is provided.

SEQ ID NO: 1:

XhoI

5 'TCGA G GGA ACT GGA CAG CGT GTC GAAA 3'

3 '

C CCT TGA CCT GTC GCA CAG CTTT AGCT 5 '

Gibco-BRL, Basel by electrophoresis

XhoI

Providing a linker end consists of the following steps. -100ng of double-stranded DNA (see SEQ ID NO: 1) was ligated to lpg Xhol-digested pPFY7 at 4 ° C for 15 hours in T4 DNA ligase (lpg; 1 unit / μΐ;

Switzerland). The ligation mixture was fractionated on a 1% agarose-Tris-acetate gel. The linear fragment containing the ligated linkers was isolated and purified with GeneClean® (Biolll, CA, USA). The DNA was again heat-treated by incubation at 95 ° C, then slowly cooled to room temperature and then transformed into E. coli strain HB100. The DNA from the transformants was analyzed on a 2% agarose gel. Clones showing strong growth after Xhol and BamHI cleavage are those that contain the UPR element in the correct orientation in the CYC1 core

-20 ··································· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · One such plasmid was named pCS2-1 (Figure 1). The insert was confirmed by DNA sequencing using Applied Biosystems DNA Sequencing 370A.

The 22bp misfolded protein response element (see SEQ ID NO: 1) is ligated to pCS2-1 after re-tagging (as described above). This results in plasmid pCS2-2, which encodes two copies of the UPR element and which is verified by DNA sequencing (see above).

The plasmids pCS2-3, pCS2-4 and pCS2-5 were similarly obtained. These encode three, four and five UPR elements, respectively. All inserts were confirmed by DNA sequencing (see above).

Example 3: Chromosomal integration of UPR-CYClp-lacz fusion genes into yeast strain W3116

Bio-Rad electroporation

Guarante, Methods-Enzymol, yeast strain (Máta HIS3 pep4-AH37) was used to produce the chromosome,

J. Biochem.

(Winston pJW1137 is used for the transformation of yeast cells (Becker &

(1991), 194, 182-187). W3116 leu2-112 his3 ura3-52 pcrl-Δ ::

Carlsberg Laboratory, Denmark)

W3116 from W3094 et al., Eur.

gene replacement

101, 211-228), a

PEP4 gene 6, 2490leu2-3 (J. Winther,

When integrating UPR-CYClp-lacz. As described in van den Hazel (1992), 207, 277-283), et al., Methods Enzymol. (1983) using a plasmid. This plasmid is a (Anmerer et al., Mol. Cell. Biol. (1986)).

2499), which is a deletion from the EcoRI site and a Clal restriction site

-21 sites, corresponding to the promoter and the first 75% of the open reading frame.

The UPR-CYClp-lacz constructs (pCS2-1 to 5) were integrated into the LEU2 locus of BstEII-cleaved W3116 after the plasmids were cleaved with BstEII. Correct gene integration and gene substitutions are confirmed by PCR. The amplified fragments were then analyzed by agarose gel electrophoresis.

Table 1: Yeast strains carrying UPR-CYClp-lacz fusion on their chromosome

Tribe Genotype UPR-CYClp-lacz fusion W3116 Mata leu2-3 leu2-112 his3 ura3- - 52 prcl-Δ :: HIS3 pep4-Á 1137 W3116-1 x lacz Mata LEU2 :: pCS2-l his3 ura3-52 1 x UPR-CYClp-lacz prcl-Δ :: HIS3 pep-4-Δ 1137 W3116-2 x lacz Mata LEU2 :: pCS2-2 his3 ura3-52 2 x UPR-CYClp-lacz prcl-Δ :: HIS3 pep4-A 1137 W3116-3 x lacz Mata LEU2 :: pCS2-3 his3 ura3-52 3 x UPR-CYClp-lacz prcl-Δ :: HIS3 pep4-A 1137 W3116-4 x lacz Mata LEU2 :: pCS2-4 his3 ura3-52 4 x UPR-CYClp-lacz prcl-Δ :: HIS3 ρβρ4-Δ 1137 W3116-5 x lacz Mata LEU2 :: pCS2-5 his3 ura3-52 5 x UPR-CYClp-lacz prcl-Δ :: HIS3 ρβρ4-Δ 1137

. Example: Faulty protein folding in the endoplasmic reticulum caused by glycosylation of a tunicamycin inhibitor and inducing the UPR-CYClp-lacz genes

-22All 1. The strain in the table is grown as a preculture in SD medium (0.67% yeast nitrogen base without amino acids, 2% glucose) for 24 hours at 30 ° C. An aliquot of the preculture (1%) was inoculated into YPD medium (1% bacto-yeast extract, 2% bacto-peptone, 2% glucose) and the cells were grown for 16 hours at 30 ° (control). To induce defective protein folding, aliquots of mid-logarithmic cultures were treated with tunicamycin (10 µg / ml; Sigma) for 6 hours at 30 ° C. Cells were harvested and washed with 0.9% NaCl. Β-galactosidase activity, as measured by hydrolysis of onitrofeml-β-D-galactopyranoside at 420 nM, is adjusted to the cell culture density and expressed in arbitrary units. The activity of the control strain (which was not treated with tunicamycin) and does not carry plasmid pPFY7 is taken as 1. The results, which are the average of the values obtained for the three individual integrals (each measurement was performed twice), are shown in Table 2.

Table 2: Induction of β-galactosidase by tunicamycin in yeast strains carrying UPRlacZ

Tribe merger β-gal β-gal activity activity (-) tunicamycin (+) Of tunicamycin W3116 - 1 1 W3116-1 x lacz 1 x UPR-CYClp-lacz 1 1.5 W3116-2 x lacz 2 x UPR-CYClp-lacz 1 3 W3116-3 x lacz 3 x UPR-CYClp-lacz 1 12 W3116-4 x lacz 4 x UPR-CYClp-lacz 1 16 W3116-5 x lacz 5 x UPR-CYClp-lacz 1 17

-235. Example: Encoding an expression cassette for wild-type CPY

Construction of a 2-micron plasmid

Plasmid pLV9, which contains the complete PRC1 gene encoding the yeast carboxypeptidase Y enzyme (CPY), was constructed as described by Valis et al., Cell (1987),

48, 887-897. Isolated from pLV9

The Clal-HindIII PRC1 fragment (containing the promoter, the preproCPY coding sequence and the transcription terminator) and a SalI-SacI fragment (5 '-> 3') were transformed into a 2 micron based pDP34 (DSM 4473) vector and For cloning into vector pFL38 (ATCC 77203). Restriction sites at the 5 'and 3' ends of PRC1 were transformed using the SalI and SacI linkers available from Boehringer.

Plasmid pDP34 is an E. coli - S. cerevísiae shuttle vector containing the entire 2 micron plasmid and encodes the URA3 and dLEU2 genes as a yeast selection marker (EP-A-34 017 0).

PFL38 is also an E. coli - S. cerevisiae commuter vector, which contains the centromere of CEN6, an autonomously replicating sequence (ARS) of S. cerevisiae, and encodes S. cerevisiae URA3 as a yeast selection marker.

After subcloning the SalI-SacI PRC1 gene fragment into pDP34 (fully digested with SalI and SacI), one of the correct plasmids was designated pDC13.

The Sall-SacI PRC1 gene fragment was also subcloned into pFL38, which was completely digested with Sall and Sacl to give pFL38 / CPY (Figure 2).

-246. EXAMPLE 1: Construction of Mutant CPY Precursor Genes Mutations inserted at the C-terminus of the CPY polypeptide sequence are shown in Table 3. The Asnlll and Lysll2 residues of the prepro-CPY sequence (shown in Table 3 as N and K single letter codes) are the sites where proteinase B converts the pro-CPY precursor to mature CPY. Amino acid residues substituted in the wild-type (wt) sequence are underlined.

Table 3: Amino acid substitutions in pro-CPY proteins mutated by PCR-mediated site mutagenesis

CPY variant The CPY pro sequence C- Used for mutagenesis terminal amino acid primers s sequence a Identification number wt (like AIENYQLRVNK - pFL38 / CPY in) mutli AIENYQKRVNK 2 and 3 Mut2 AIENLDKRVNK 4 and 3 Operation 3 AIENYKRDPGK 5 and 3

Mutations are made by PCR-mediated site-directed mutagenesis of a 157 bp Munl-BamHI fragment of PRC1 contained in pFL38 / CPY (Wals et al., Cell (1987), 48, 887-897). The primers used in the PCRs are shown in Table 3.

A SalI-MunI fragment of pFL38 / CPY (see Figure 2 and Example 5) and the mutated MunI-BamHI fragment (see

ί 1

-25 · · · · above) was first subcloned into fully digested pUC19 with SalI and BamHI. The resulting plasmids contain a 1159 bp SalI-BamHI fragment which is the 5 'end of PRC1. Mutations are confirmed by DNA sequencing (see Example 2).

Example 7: Construction of 2-micron plasmids encoding expression cassettes of mutant CPY precursor genes

The complete mutant genes encoding the promoter, coding sequence and transcription terminator are assembled as SallSacI fragments.

One SalI-BamHI fragment (containing a mutant sequence of CPY and subcloned into pUC19, see Example 7), one BamHI-NcoI (931 bp) and one NcoI-SacI fragment (546 bp) (the latter two fragment from pFL38 / CPY (see Figure 1 and Example 5) was subcloned into a fully digested pDP34 with SalI and BamHI. The resulting plasmids were named pDC8 (encoding Mut1 PRCl), pDC9 (encoding Mut2 PRC1t), and pDCIO (encoding Mut3 PRCl). They are used for expression in yeast.

Example 8: Mutant PRC1 genes express misfolded proteins

PDC8 (encodes Mutl PRCl), pDC9 (encodes Mut2 PRCl), pDCIO (encodes Mut3 PRCl) and pDC13 (encodes wt PRCl) are transformed into W3116-3 x lacz by electroporation (see Figure 3). example). The β-galactosidase expression induced by the mutant PRC1 genes is compared to strains carrying the wild-type gene (wt gene). Three individual transformants of each of the above four transformations were grown as described in Example 4. Each essay is twice

-26 we're done. The results are shown in Table 4. The β-galactosidase activity in strain YDC13, which expresses wt PRCl, is taken as].

Table 4: Induction of β-galactosidase by mutated pro-CPY molecules in a strain carrying 3 x UPR-lacz

plasmid PRC1 allele Yeast transformant β-gal activity pDC13 wt YDC13 1 pDC8 mutli YDC8 3 pDC9 Mut2 YDC9 4 PdCl Operation 3 YDC10 2.5

Similar results were obtained with strain W3116-4 x lacz.

Example 9: Mutated pro-CPY proteins can also be shown to be flawed when the lacz reporter gene is regulated by the entire KAR2 promoter

To construct the KAR2p-lacZ construct, an XhoI-BamHI fragment containing the CYC1 promoter was removed from the plasmid pCS2-1 and a 630 bp

Sall-BamHI is replaced by the KAR 2 promoter fragment (Rose et al., Cell (1989) 57, 12223-1236). The KAR2 promoter was isolated by PCR using two primers having SEQ ID NOs: 6 and 7. The template for PCR is the yeast strain S288C (ATCC 26108). The plasmid thus obtained is named pCSFOL4. This was integrated into W3116 as described in Example 3. One of these strains is called W3116KAR2p-lac.

Plasmid pDC8 (encodes Mut1 PRCl), pDC9 (encodes Mut2 PRCl), pDCIO (encodes Mut3 PRCl) and pDC13 (encodes wt PRCl) were transformed into strain W3116KAR2p-lacz (see Figure 3). example).

The β-galactosidase expression induced by the mutant PRC1 genes is compared with strains carrying the wt gene. Three individual transformants of each of the above four transformations were grown as described in Example 4. Each essay is performed twice. The results are shown in Table 5. The β-galactosidase activity of the strain transformed with pDC13 encoding wt PRCl is taken as 1.

Table 5: Induction of β-galactosidase by mutated pro-CPY molecules in a strain carrying KAR2p-lacz

plasmid PRC1 allele β-gal activity pDC13 wt 1 pDC8 mutli 1.5 pDC9 Operation 2 2 PdCl Mut3 1.2

Example: Construction of an expression cassette for human β-amyloid peptide (βΑ4) without signal peptide

Plasmid pJC21 (which encodes only fiA4 without the signal sequence; expression is regulated by the GAPCL promoter) is constructed as follows. A fragment of the GAPCL promoter, a SAlI-BglII fragment (consisting of a -275 bp SallBamHI pBR322 fragment and a -400 bp yeast GAPDH gene ··· ·

-28 containing the promoter fragment) is isolated by PCR using primers having SEQ ID NOs: 8 and 9. As template, plasmid pBCl was prepared as described by Chaudhuri et al., Eur. J. Biochem. (1992), 206, 793-800.

The double-stranded DNA encoding the 42 amino acid βΑ4 peptide (Müller-Hill & Beyreuther, Annu. Rev. Biochem. (1989) 58: 287-307) is chemically synthesized according to the method described in 10./11. using oligomers having SEQ ID NO: 12 and PCR using two primers (SEQ ID NOs: 12 and 13). The BglII-EcoRI fragment of β4 was subcloned into pUC19BgI (the BamHI site of pUC19 was modified to BglII to obtain pUC19BgI; the BamHI site was filled in with Klenow polymerase; sequence) and the sequence is verified as described in Example 2.

10./11. SEQ ID NO:

BglII Met

5 'AGATCTGATG GACGCTGAAT TTAGACACGA CTCTGGTTAC GAAGTTCACC

ACCAAAAGTT GGTCTTCTTC GCTGAAGACG TTGGT 3 '(Section 10)

s zeq)

3 'CCAGAAGAAG CGACTTCTGC AACCAAGATT GTTCCCACGA

TAATAACCAA ACTACCAACC ACCACAACAC TAGCGAATTC TTAAG 5 '(Seq. 11)

Eco

From the yeast SUC2 gene (Taussing & Carlson, Nucleic Acids Res. (1983), 11, 1943-1954), an EcoRI-SacI terminator · · · · · · · · · · · · · · · · · · · ·

-29 using PCR using available primers. Yeast genomic DNA (from wild-type strain S288C) was used as a template.

Three fragments ((SalI-BglII, BglII-EcoRI and EpoRI-SacI) were subcloned into pDP34 which was completely digested with SalI and SacI to give pJC21 (see Figure 6).

Table).

Example: Construction of human β-amyloid peptide (βΑ4) expression cassettes containing signal peptide

PJC22 (encodes the SUC2 signal sequence linked to 3A4; expression under the control of the GAPCL promoter), pJC26: (encodes the βC4-ύθζ linked PRC1 preproquence;

expression by control of the PRC1 promoter) and pDP34-NLSβΑ4-ό (encoding the nucleus localization sequence from SV40 large T antigen, NLS and PA4; expression under the control of the GAPCL promoter) were constructed similarly to pJC21. (see Example 10; 6.

Table).

The SalI-BglII fragment containing the GAPCL promoter linked to the SUC2 signal sequence (i.e., pBR322-GAPCLp-Invss) was amplified by PCR using primers having SEQ ID NOs: 8 and 16. Plasmid pBCl (see Example 10) was used as template.

A Sall-BglII fragment containing the PRC1 promoter linked to the CPY prepro sequence (i.e., CPYp-prepro) was amplified by PCR using primers having SEQ ID NOs: 17 and 18, template pLV9 (see Examples 5 and 6). .

4> »··· ·

-30A Sal-BglII fragment (i.e. pBR322-GAPCLp-NLS) containing the GAPCL promoter linked to the nucleus localization sequence of the SV40 T antigen was amplified by PCR using primers having SEQ ID NOs: 8 and 19. The PCR template is plasmid pRH3, which encodes a -275 bp SalI-BamHI fragment from pBR322, encodes a 400 bp BamHI-EcoRI GAPCLp fragment, and encodes an EcoRI-Spel fragment of the SV40 T antigen nuclear localization sequence. .This nucleus localization sequence

(Than salt & Si 1 ver , Mo 1. Cell. Biol. ( ; 198 9) 9, 384-389) synthesized the 30 SEQ ID NO: s sequence deoxy oligo oribonucleotide. 30th SEQ ID NO: sequence: Eco M D K V F R N S S R T P P 5 ' AATTC ATG GAC AAG GTC TTC AGA AAC TCT TCC AGA ACT CCA CCA 3 ' G TAC CTG TTC CAG AAG TCT TTG AGA AGG TCT TGA GGT GGT K K K R K V E D P THE AAG AAG AAG AGA AAG GTT GAA GAC CCA GCA 3 ' TTC TTC TTC TCT TTC CAA CTT CTG GGT CGT GATC 5 '

Spe

One of the above Sall-BglII fragments, the BglIII-EcoRI fragment (encoding pA4, see Example 10) and the EcoRISacI fragment (encoding the SUC2 terminator, see Example 10), were subcloned into pDP34 ( completely digested with Sallgy and Sacl) to give pJC22, pJC26 and pDP34ΝΒΞ-βΑί-Ι.

* · »···

-316. Table: βΑ4 expression cassettes with and without signal peptide

plasmid name expression cassette Fragments of which it is composed 1ítottuk pJC21 GAPCLp ^ A4- Sall-BglII GAPCLp + BglII -EcoRI βΑ4 SUC2 (chemically synthesized) + SUC2 EcoRI-SacI pJC22 GAPCLp-invss- Sall-BglII GAPCLp-invss + EcoRI-BglII flA4-SUC2 βΑ4 (chemically synthesized SacI SUC2t + EcoRI- pJC2 6 CPYp-preproCPY- Sall-BglII CPYp-preproCPY + EcoRI-BglII flA4-SUC2 βΑ4 (chemically synthesized SacI SUC2t + EcoRI- pDP34- GAPCLp-NLS- βΑ4- Sall-BglII GAPCLp-invss + EcoRI-BglII NLS βΑ4 SUC2 βΑ4 (chemically synthesized SacI SUC2t + EcoRI-

Example 12: Yeast transformation with pJC21, pJC22, pJC26 and pDP34-NLS-PA4, and the β-galactosidase assay for the transformants

The plasmids were transformed into W3116-3 x lacz (see Example 3). Β-galactosidase activity was measured (as in Example 4), see Table 7.

· * ** ·· «··· • · *« · «9 *« ·. · Β • ·· · * ·> ·

-327. Table: Only βΑ4 in the secretory pathway exhibits defective folding in the UPR-lacZ assay

Name of the plasmid expression cassette W3116-3xlacz transformed β-gal activity pJC21 GAPCLp-PA4-SUC2 YJC21 1 pJC22 GAPCLp-invss- PA4-SUC2 YJC22 3 pJC2 6 CPYp-preproCPY- PA4-SUC2 YJC2 6 4 pDP34-NLS- βΑ4 GAPCLp-NLS- βΑ4- SUC2 3 x lacz-NLS- βΑ4 1

Example 13: C-terminal truncations of βΑ4; yeast transformation; β-galactosidase assay

Two plasmids, pJC24 (encodes the SUC2 signal sequence and a truncated version of βΑ4, 1-39) and pJC25 (encodes the SUC2 signal sequence and a truncated version of βΑ4, 1-36) as described below. The aggregation of 1-3 9 βΑ4 and 1-3 6 βΑ4 is lower in vitro (Burdick et al., J. Bioi. Chem. (1992), 267, 546-554; Barrow et al., Mol. Bioi. (1992)). 225, 10751093; C. Pike et al., J. Neuroscience (1993) 13, 1676-1687).

Two SalI-XbaI fragments, pBR322-GAPCLp-Invss ^ A4 1-39 and pBR322-GAPCLp-Invss ^ A4 1-36 were isolated by PCR using two primers (SEQ ID NOs: 8 and 20). the previously mentioned fragment and SEQ ID NOs: 8 and 21 in the latter construct). The template is pJC22. The Sall-Xbal fragment and the Xbal-SacI:, ·· · * ··· • * · * · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

The -33 fragment was subcloned into pDP34, which was completely cleaved with SalI and SacI (see Table 8).

Table 8. Expression cassettes for C-terminal truncated versions of βΑ4

plasmid Expression cassette Fragments of which name We were pJC24 GAPCLp-invss ^ A4 (1- Sall-Xbal GAPCLp-invss-βΑ4 1-39 {PCR 39) -SUC2t product) + Xbal-SacI SUC2t pJC25 GAPCLp-Invss-βΑ4 (1- Sall-Xbal GAPCLp-invss-βΑ4 1-36 {PCR 36) SUC2 product) + Xbal-SacI SUC2t

The plasmids were transformed into strain W3116-3 x lacz (see Example 3). Β-galactosidase activity was measured (similar to Example 4) and compared to YJC21 and YJC22 strains described in Example 10 (see Table 9).

Table 9: C-terminally truncated βΑ4 molecules have less hair loss than wt βΑ4 in the UPR-lacz assay

Name of the plasmid expression cassette W3116-3xlacz transformed β-gal activity pJC24 GA P C Lp-1nvs s- βΑ4 (1-39) -SUC2t YJC2 4 1.8 p JC25 GAPCLp-invs s- βΑ4 (1-36) SUC2t YJC25 2.2 pJC21 GAPCLpLA4-SUC21 YJC21 1 pJC22 GAPCLp-invss- 3A4 SUC2 YJC22 3

• · • · · ·

-3 414. Example: Rat mutant βΑ4 (mr βΑ4) and inverted human βΑ4 (ίβΑ4)

Plasmid pJC27 and pJC28 were constructed as shown in Table 10. Both expression cassettes contain the prepro sequence of the PRC1 gene and are directed by the PRC1 promoter. PJC27 expresses the rat mutant βΑί (mrPA4) (Dyrks et al., FEBS Lett. (1993) 324, 231-236; Hilbrich et al., Mol. Biol. (1991) 218, 149-163) and pJC28 is the inverted human βΑ4-ί (ίβΑ4) (Fraser et al., Biochemistry (1992), 31,

10716-10723).

As described in Table 10, the plasmids were derived from a SalI-BglII fragment (containing the CPY promoter and the CPY prepro sequence), a BglII-EcoRI fragment (containing either m ^ A4 or ßβ4-ό) and SUC2. terminator is constructed from an Eco RI-Sac I fragment.

The BglII-EcoRI fragments (either containing τητβΑί-ί or ίβΑ4) are two overlapping oligodeoxyribonucleotides (see

25./26. and 27/28. SEQ ID NO: 12) and two primers (see SEQ ID NOS: 12 and 13) which hybridize to the 5 'and 3' ends of the fragments.

The SalI-BglII, BglII-EcoRI and EcoRI-SacI fragments were subcloned into pDP34 completely cleaved with SalI and SacI.

• ·

-3 510. Table: βΑ4 expression cassettes for rat mutant βΑ4 mrfiA4) and inverted human βΑ4 (ifiA4)

plasmid name Expression cassette Fragments of which compiled pJC2 7 CPYp-mr-preproCPY Sall-BglII CPYp-preproCPY (11 · fiA4-SUC2 example) + BglII-EcoRI mr βΑ4 (a 23/24, 12, 13 oligomers of SEQ ID NO applying Synthesized by PCR) + EcoRI-SacI SUC2t (Example 10) pJC2 8 CPYp-preproCPY-βΑ4- Sall-BglII CPYp-preproCPY {11. SUC2 Example 1) + BglII-EcoRI τβΑ4 (a 25/26, 12, 13 oligomers of SEQ ID NO applying Synthesized by PCR) + EcoRI-SacI SUC2t (Example 10)

The amino acid sequences of βΑ4, mrfiA4 and ίβΑ4 are shown in Figure 11.

This is shown in the table. The differences from the human β hum4 sequence of wt are underlined.

23./24. SEQ ID NO:

BglII Met

5 'AGATCTGATG GACGCTGAAT TTGGTCACGA CTCTGGTTTC GAAGTTAGAC

ACCAAAAGTT GGTTATCGGT GCTGAAGACG TTGGT 3 '(Seq. 23)

3 'CCAATAGCCA CGACTTCTGC AACCAAGGTT GTTCCCACGA

TAGTAACCAA ACTACCAACC ACCACAACAG TAGCGAACTC TTAAG 5 '(SEQ ID NO: 24)

EcoRI ·· · · · · · · · · · · · · · · · ·

25./26. SEQ ID NO: BglII Met 5 'AGATCTGATG GCTATCGTTG TCGGTGGTGT TATGTTGGGT ATCATTGCTG GTAAGAACTC TGGTGTTGAC GAAGCTTTCT TCGTT 2 '( 2 5. seq.) 3 ' ACCACAACTG CTTCGAAAGA AGCAAAACTT CGTTGTGGTG

CAACTTATGC CAAGACTGGT GTCTAAGCTT CGACTGACTC TTAAG 5 '(SEQ ID NO: 26) EcoRI

Table 11: βΑ4, ιηΓβΑ4 and ίβΑ4 peptide sequences (27,

SEQ ID NOs: 28 and 29) βΑ4 DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGW IA

SEQ ID NO: 27

ΓΠΓβΑ4 DAEFGHDSGF EVRHQKLVIG AEDVGSNKGA IIGLMVGGW IA

SEQ ID NO: 28 ιβΑ4 AIVVGGVMLG IIAGKNSGVD EAFFVLKQHH VEYGSDHRFE AD

SEQ ID NO: 29

The plasmids in Table 10 were transformed into strain W3116-3 x lacz (see Example 3). The β-galactosidase activity was measured as described in Example 4 and compared to YJC26, YJC21 and YJC22 strains described in Example 10 (see Table 12).

• · · ·

-3 712. Table: Rat mutant βΑ4 (mr3A4) and human βΑ4 (ίβΑ4) βΑ4 molecules do not malform in UPR-lacZ assay

plasmid name Expression cassette W3116-3xlacz transformed β-gal activity pJC27 CPYp-preproCPY- mrfiA4-SUC2 YJC27 1.2 pJC2 8 CPYp-preproCPY- 1βΑ4-Ξυα21 YJC28 1 pJC2 6 CPYp-preproCPY-β- A4 SUC2 YJC2 6 4 _P JC22 GAPCLp-Invss-PA4- SUC2 YJC22 3 pJC21 GAPCLp-fiA4-SUC2 YJC21 1

Example 15: Trivalent Aluminum and Divalent Zinc Increase Defective Folding of βΑ4 in Yeast

It is known that Al es

Zn induces βΑ4 aggregation in vitro (Mantyh et al., J. Neurochemistry (1993) 61, 1171-1174). Β-galactosidase activity was measured as in Example 4 after cells were grown in YPD with AlCl 3 (~ 3mM; Merck) and ZnCl 3 (~ 300µm;

Merck). The results are shown in Table 13. The -s control symbol indicates that cells were grown without both transition metal cations.

• · ·

Zn ²⁺ induces better in βΑ4 variants

-3813. Table hair twitching, 3+

Al and a in a 3A4 like the bug

plasmid expression W3116-3xlacz β-D-GA β-GA β-GA name Cassette transformed control + A1C1 ₃ + ZnCl ₂ pJC26 CPYp-preproCPY- b-A4 SUC2 YJC2 6 4 9 5, 5 pJC22 GAPCLp-invss- βΑ4-Ξυθ2ί YJc22 3 7 4 pJC21 GAPCLp ^ A4 SUC2 YJC21 1 1.6 1.1 pJC24 GAPCLp-invss- βΑ4 (1-39) -SUC2t YJC24 1, 8 2 1.5 p JC2 5 GAPCLp-invss- βΑ4- (1-36) SUC2 YJC25 2.2 3.5 2.7 pJC27 CPYp-preproCPY- m ^ A4 SUC2 YJC27 1.2 1.5 no because pJC2 8 CPYp-preproCPY- 1βΑ4-3υ02Ρ YJC2 8 1 1.3 no because

Example 16: Desferal and Ascorbic Acid Decrease Defective Folding Inhibition of βΑ4

ΒΑό is expressed in minimal SD medium (see Example 4) in the presence or absence of desferrioxamine (Desferal® CYBA-GEIGY) and ascorbic acid (Merck). Β-galactosidase activity was measured as described in Example 4. The -s control symbol indicates that the cells were grown without desferrioxamine or ascorbic acid.

• · ·

-3 914. Table: Desferal prevents βΑ4 from folding incorrectly

plasmid expression W3116-3xlacz β-D-GA β-GA + β-GA + name Cassette trans- control Desferal Desferal 4, 5µΜ formant 1 4,5μΜ + Fe ^{J +} 5µΜ pJC2 6 CPYp- YJC2 6 4 1.4 3, 5 preproCPY-b- A4 SUC2 pJC21 GAPCLp-bA4- YJC21 1 1 1 SUC2

Table 15: Ascorbic acid also prevents βΑ4 defective hair

plasmid expression W3116-3xlacz b-D-GA b-GA aces- name cassette transformed control binary acid ImM pJC26 CPYp-preproCPY- b-A4 SUC2 YJC2 6 4 1, 9 pJC21 GAPCLp-bA4- SUC2 YJC21 1 1

Both DFO and ascorbic acid appear to reduce defective folding / aggregation of βΑ4 in yeast cells.

Deposits

The following microorganism strain was deposited with the Deutsche Sammlung von Mikroorganismen (DSM), Mascheroder Weg lb, D-38124 Braunschweig (with registration number and date of deposit):

E. coli JM109 / pDP34 DSM4473

March 14, 1988

* ί ·· · ·

-40 Sequence Listing (1) General Information:

(i) Being contrary to:

(A) Name: Ciba-Geigy AG.

(B) Street: Klybeckstr. 141st

(C) City: Basel (E) Country: Switzerland (F) Postcode (ZIP): 4002 (G) Phone: +41 61 69 11 11 (H) Fax: + 41 61 696 79 76 (I) Telex: 962 991 (ii) Title of the Invention: Method for Detecting False Folded Proteins (iii) Number of Sequences: 30 (iv) Computer Processing Form:

(A) Media Type: Floppy Disk (B) Computer: IBM PC Compatible (C) Operating System: PC-DOS / MS-DOS (D) Software: Patentln Release # 1.0,

Version # 1.25 (EPO) (vi) Earlier Announcement Details:

(A) Filing No. 94810536.6 (B) Filing Date: Sep 1994 16th

(2) Information for SEQ ID NO: 1:

(i) Sequence characteristics:

(A) Length: 31 base pairs (B) Type: nucleic acid

·· · ·

-41 (C) Fiber type: double-stranded (D) Topology: linear (ii) Molecular type: DNA (genomic) (ix) Characteristics:

(A) Name / Key: RNA Mixed (B) Location: 5..27 (D) Other Information: / Product = UPR Element (ix) Features:

(A) Name / Key: Mixed Feature (B) Location: 1..4 (D) Other Information: / Function = Xhol Linker (ix) Features:

(A) Name / Key: Mixed Feature (B) Location: 28..31 (D) Other Information: / Function = Xhol Linker (xi) Sequence Description: SEQ ID NO: 1:

TCGAGGGAAC TGGACAGCGT GTCGAAATCG A 31 (2) Information for SEQ ID NO: 2:

(i) Sequence characteristics:

(A) Length: 45 base pairs (B) Type: nucleic acid (C) Fiber type: single-stranded (D) Topology: Linear (ii) Molecular type: DNA (genomic) (ix) Characteristics:

(A) Name / Key: Mixed Feature (B) Location: 1..45 • · · · + 1

-42 (D) Other information: / function = primer for site-directed mutagenesis / note = intelligent strand of the yeast PRC1 gene for PCR reaction / (ix)

(A) Name / Key: Mixed Feature (B) Location: 4..9 (C) Other Information: / Function = MunI Location (ix) Features:

(A) Name / Key: Mixed Feature (B) Location: 21..23 (D) Other Information: / Function = Mutation versus Wild Type (xi) Sequence Description: SEQ ID NO: 2:

ACGCAATTGA AAACTATCAG AAGCGTGTCA ACAACAAGAT TAAGG 45 (2) Information for SEQ ID NO: 3:

(i) Sequence) Opponents:

(A) Length: 27 base pairs (B) Type: nucleic acid (C) Fiber type: single-stranded (D) Topology: linear (ii) Molecular type: DNA (genomic) (ix) Characteristics:

(A) Name / Key: Mixed Feature (B) Location: Complementary (1..27) (C) Other Information: / function = PCR primer / 'note = antisense strand of yeast PRC1 gene for PCR reaction

43 (ix) Features:

(THE) Name / Key: Mixed specific (B) Location: 5 .. . 10 (D) Other informations: : / function = BamHI space

(xi) Sequence description: SEQ ID NO: 3:

GACCGGATCC TTTGCAGGAT CGTTTCT 27 (2) Information for SEQ ID NO: 4:

(i) Sequence markers:

(A) Length: 41 base pairs (B) Type: nucleic acid (C) Fiber type: single-stranded (D) Topology: linear (ii) Molecular type: DNA (genomic) (ix) Characteristics:

(A) Name / Key: Miscellaneous (B) Location: 1..41 (D) Other information: / function = primer for site-directed mutagenesis / note = sense strand of yeast PRC1 gene for PCR reaction (ix)

(A) Name / Key: Mixed Feature (B) Location: 4..9 (D) Other Information: / Function = MunI Location (ix) Features:

(A) Name / Key: Mixed Feature (B) Location:

group (16.19, 21.24)

-44 (D) Other information: / function = mutation relative to wild type (xi) Sequence description: SEQ ID NO: 4:

ACGCAATTGA AAACTTGGAC AAGCGTGTCA ACAACAAGAT T 41 (2) Information for SEQ ID NO: 5:

(i) Sequence characteristics:

(ii) (ix)

IX (ix)

XI (A) Length: 47 base pairs (B) Type: nucleic acid (C) Fiber type: single-stranded (D) Topology: linear

Molecule Type: DNA (Genomic)

Distinctive features:

(A) Name / Key: Mixed Feature (B) Location: 1..47 (D) Other Intrinsic Information: / Function = Primer for Site-directed Mutagenesis "/ Note = Sensory strand of yeast PRC1 gene for PCR reaction

Distinctive features:

(A) Name / Key: Mixed Feature (B) Location: 4..9 (D) Other Information: / Function = MunI Location Features:

(A) Name / Key: Mixed Feature (B) Location: Group (18, 22, 24..32) (D) Other Information: / Function = Mutation versus Wild Type

Sequence description: SEQ ID NO: 5:

• · ····

-45ACGCAATTGA AAACTATAAG CGTGACCCAG GTAAGATTAA GGACCCT 47 (2) Information for SEQ ID NO: 6:

(i) Sequence characteristics:

(A) Length: 28 base pairs (B) Type: nucleic acid (C) Fiber type: single-stranded (D) Topology: linear (ii) Molecular type: DNA (genomic) (ix) Characteristics:

(A) Name / Key: Mixed Feature (B) Location: 1..28 (D) Other Information: / Function = PCR Primer / Note ^ Sense strand of the yeast KAR2 gene promoter (ix) Features:

(A) Name / Key: Mixed Feature (B) Location: 3..8 (D) Other Information: / Function = Scarf Location (xi) Sequence Description: SEQ ID NO: 6:

TAGTCGACAC TTCAATGTCT AATGCTAG 28 (2) Information for SEQ ID NO: 7:

(i) Sequence characteristics:

(A) Length: 25 base pairs (B) Type: nucleic acid (C) Fiber type: single-stranded (D) Topology: linear (ii) Molecular type: DNA (genomic) d e 1 1 e g e

(ix) • ·· · ·· · ·

-4 6 (A) Name / Key: Mixed Feature (B) Location: Complementary (1..25) (D) Other Information: / Function = PCR primer / Note = antisense strand of the yeast KAR2 gene promoter (ix)

(A) Name / Key: Mixed Feature (B) Location: 3..8 (D) Other Information: / Function = BamHI Location (xi) Sequence Description: SEQ ID NO: 7:

TAGGATCCGG TATGTTTGAT ACGCT 2 (2) Information for SEQ ID NO: 8:

(i) Sequence Opponents:

(A) Length: 28 base pairs (B) Type: nucleic acid (C) Fiber type: single-stranded (D) Topology: linear (ii) Molecular type: DNA (genomic) (ix) Characteristics:

(A) Name / Key: Mixed Feature (B) Location: 1..28 (D) Other Information: / Function = PCR Primary / Note = sense strand of pBR322 (ix) Features:

(A) Name / Key: Mixed Feature (B) Location: 3..8 (D) Other Information: / Function = Scarf Location (xi) Sequence Description: SEQ ID NO: 8:

• · «···

-47TAGTCGACGC TCTCCCTTAT GCGACTCC 28 (2) Information for SEQ ID NO: 9:

(i) Sequence characteristics:

(A) Length: 28 base pairs (B) Type: nucleic acid (C) Fiber type: single-stranded (D) Topology: linear (ii) Molecular type: DNA (genomic) (ix) Characteristics:

(A) Name / Key: Mixed Feature (B) Location: Complementary (1..28) (D) Other Information: / Function = PCR primer / Note = antisense strand of the yeast GAPDH gene promoter sequence (ix)

(A) Name / Key: Mixed Feature (B) Location: 3..8 (D) Other Information: / Function = BglII Location (xi) Sequence Description: SEQ ID NO: 9:

ATAGATCTTT TGTTTATGTG TGTTTATT 28 (2) Information for SEQ ID NO: 10:

(i) Sequence characteristics:

(A) s o s: 8 5 b a z pairs (B) Type: nucleic acid (C) Fiber type: single-stranded (D) Topology: linear (ii) Molecular type: DNA (genomic) (ix) Characteristics:

··

-48 (A) Name / Key: Mixed Feature (B) Location: 1..85 (D) Other Inserts: / Function = DNA with Sticky Ends / Note = Human BetaA4 Peptide Sense (ix) Features:

(A) Name / Key: Mixed Feature (B) Location: 1..6 (D) Other Information: / Function = BglII Location (ix) Features:

(A) Name / Key: Mixed Feature (B) Location: 61..85 (D) Other Information: / Function = Overlapping with 11.

(xi) Sequence description: SEQ ID NO: 10:

AGATCTGATG GACGCTGAAT TTAGACACGA CTCTGGTTAC GAAGTTCACC 50

ACCAAAAGTT GGTCTTCTTC GCTGAAGACG TTGGT 85 (2) Information for SEQ ID NO: 11:

(i) Sequence characteristics:

(A) Length: 85 base pairs (B) Type: nucleic acid (C) Fiber type: single-stranded (D) Topology: linear (ii) Molecular type: DNA (genomic) (ix) Characteristics:

(A) Name / Key: Mixed Feature (B) Location: Complementary (1..85)

• · ·· · ·

-49 (D) Other information: / function = other part of DNA with sticky ends / note = antisense strand of human betaA4 peptide (ix)

(A) Name / Key: Mixed Feature (B) Location: 79..85 (D) Other Information: / Function— EcoRI Location (ix) Features:

(A) Name / Key: Mixed Feature (B) Location: 1..25 (D) Other Information: / Feature = Overlapping 10.

(xi) Sequence description: SEQ ID NO: 11:

CCAGAAGAAG CGACTTCTGC AACCAAGATT GTTCCCACGA TAATAACCAA

ACTACCAACC ACCACAACAC TAGCGAATTC TTAAG (2) Information for SEQ ID NO: 12:

(i) Sequence characteristics:

(A) Length: 19 base pairs (B) Type: nucleic acid (C) Fiber type: single-stranded (D) Topology: linear (ii) Molecular type: DNA (genomic) (ix) Characteristics:

(A) Name / Key: Mixed Feature (B) Location: 1..19 (D) Other Information: / Function = PCR primer »· ····

-50 / note = human betaA4 sense strand (i x)

(A) Name / Key: Mixed Feature (B) Location: 3..8 (D) Other Information: / Function = BglII Location (xi) Sequence Description: SEQ ID NO: 12:

ATAGATCTGA TGGACGCTG 19 (2) Information for SEQ ID NO: 13:

(i) Sequence] characteristics:

(A) Length: 20 base pairs (B) Type: nucleic acid (C) Fiber type: single-stranded (D) Topology: linear (ii) Molecular type: DNA (genomic) (ix) Characteristics:

(A) Name / Key: Mixed Feature (B) Location: Complementary (1..20) (D) Other Information: / Function = PCR Primary / Note = Antisense (ix) of Human BetaA4 Peptide:

(A) Name / Key: Mixed Feature (B) Location: 3..8 (D) Other Information: / Function = EcoRI Site (xi) Sequence Description: SEQ ID NO: 13:

ATGAATTCTC AAGCGATGAC 20 (2) Information for SEQ ID NO: 14:

·· «··«

-51 (i) Sequence Sequence j) e.

(A) Length: 28 base pairs (B) Type: nucleic acid (C) Fiber type: single-stranded (D) Topology: linear (ii) Molecular type: DNA (genomic) (ix) Characteristics:

(A) Name / Key: Mixed Feature (B) Location: 1..28 (D) Other Inner Actions: / Function = PCR Primary / Note = Sensory Fiber of the Yeast SUC2 Gene (ix) Characteristics:

(A) Name / Key: Mixed Feature (B) Location: 3..8 (D) Other Information: / Function = EcoRI Site (xi) Sequence Description: SEQ ID NO: 14:

ATGAATTCAG GTTATAAAAC TTATTGTC 28 (2) Information for SEQ ID NO: 15:

(í) Sequence markers:

(A) Length: 26 base pairs (B) Type: nucleic acid (C) Fiber type: single-stranded (D) Topology: linear (ii) Molecular type: DNA (genomic) (ix) Characteristics:

(A) Name / Key: Mixed Feature (B) Placed :: Complementary (1..26)

-52 (D) Other information: / function = PCR primer / note = antisense strand of the yeast SUC2 gene (ix)

(A) Name / Key: Mixed Feature (B) Location: 3..8 (D) Other Information: / Function = SacI Site (xi) Sequence Description: SEQ ID NO: 15:

TAGAGCTCGG TCCATCCTAG TAGTGT 26 (2) Information for SEQ ID NO: 16:

(i) Sequence markers:

(A) Length: 30 base pairs (B) Type: nucleic acid (C) Fiber type: single-stranded (D) Topology: linear (ii) Molecular type: DNA (genomic) (ix) Characteristics:

(A) Name / Key: Mixed Feature (B) Location: Complementary (1.30) (D) Other Information: / Function = PCR primer / Note = antisense strand of yeast SUC2 signal sequence

(ix) Features: (A) Name / Key: Mixed j analyst (B) Location: 3 .. . 8 (D) Other information: : / function = BglII space (xi) Sequence description: 16. SEQ ID NO

ATAGATCTGC TGCAGATATT TTGGCTGCAA • ·

-53 (2) Information for SEQ ID NO: 17:

(i) Sequence characteristics:

(A) Length: 28 base pairs (B) Type: nucleic acid (C) Fiber type: single-stranded (D) Topology: linear (ii) Molecular type: DNA (genomic) (ix) Characteristics:

(A) Name / Key: Mixed Feature (B) Location: 1..28 (D) Other Information: / Function = PCR primer / Note = Sense strand of the yeast PRC1 gene promoter (ix)

(A) Name / Key: Mixed Feature (B) Location: 3..8 (D) Other Information: / Function = Scarf Location (xi) Sequence Description: SEQ ID NO: 17

ATGTCGACAT CGATTTCCGT ATATGATG (2) Information for SEQ ID NO: 18:

(i) Sequence characteristics:

(A) Length: 30 base pairs (B) Type: nucleic acid (C) Fiber type: single-stranded (D) Topology: linear (ii) Molecular type: DNA (genomic) (ix) Characteristics:

(A) Name / Key: Mixed Feature • · · · «

-54 (B) Location: complementary (1.30) (D) Other information: / function = PCR primer / meg] match = antisense strand of the yeast PRC1 gene (ix)

(A) Name / Key: Mixed Feature (B) Location: 3..8 (D) Other Information: / Function = BglII Location (xi) Sequence Description: SEQ ID NO: 18:

ATAGATCTTT AATCTTGTTG ACACGAAGCT 30 (2) Information for SEQ ID NO: 19:

(i) Sequence markers:

(A) Length: 29 base pairs (B) Type: nucleic acid (C) Fiber type: single-stranded (D) Topology: linear (Irish) Molecular type: DNA (genomic) (ix) Characteristics:

(A) Name / Key: Mixed Feature (B) Location: Complementary (1..29) (D) Other Information: / Function = PCR primer / Note antisense strand of the SV40 T antigen nuclear localization signal (ix)

(A) Name / Key: Mixed Feature (B) Location: 3..8 (D) Other Information: / Function = BglII Location (xi) Sequence Description: SEQ ID NO: 19:

• · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · Reg ·ics ·

-55ATAGATCTGC TGGGTCTTCA ACCTTTCTC (2) Information for SEQ ID NO: 20:

(i) Sequence characteristics:

(A) Length: 29 base pairs (B) Type: nucleic acid (C) Fiber type: single-stranded (D) Topology: linear (ii) Molecular type: DNA (genomic) (i x) Characteristics:

(A) Name / Key: Mixed Feature (B) Location: Complementary (1..29) (D) Other Intrinsic Information: / Function = PCR Primary / Note = Human BetaA4 Antisense Fiber (ix) Features:

(A) Name / Key: Mixed Feature (B) Location: 3..8 (D) Other Information: / Function = Xbal Location (xi) Sequence Description: SEQ ID NO: 20:

ATTCTAGATT AAACACCACC AACCATCAA 29 (2) Information for SEQ ID NO: 21:

(í) Sequence characteristics:

(A) Length: 29 base pairs (E) Tίa jacket: nucleic acid (C) Fiber type: single-stranded (D) Topology: linear (ii) Molecular type: DNA (genomic) (ix) Characteristics:

-5 β · · · · · · · · · · · · · · · · · · · · · · · · · (A) Name / Key: Mixed Feature (B) Location: Complementary (1..29) (D) Other Information: / Function = PCR primer / Comment on human beta antisense fiber (ix)

(A) Name / Key: Mixed Feature (B) Location: 3..8 (D) Other Information: / Function = Xbal Location (xi) Sequence Description: SEQ ID NO: 21

ATTCTAGATT AAACCATCAA ACCAATAAT 29 (2) Information for SEQ ID NO: 22:

(i) Sequence characteristics:

(A) Length: 28 base pairs (B) Type: nucleic acid (G) Fiber type: single-stranded (D) Topology: linear (ii) Molecular type: DNA (genomic) (ix) Characteristics:

(A) Name / Key: Mixed Feature (B) Location: 1..28 (D) Other Innformations: / Function = PCR Primary / Note = Sensory Area of the Yeast SUC2 Gene (ix) Characteristics:

(A) Name / Key: Mixed Feature (B) Location: 2..8 (D) Other Information: / Function = Xbal Location

-57 (xi) SEQ ID NO: 22:

ATTCTAGAAG GTTATAAAAC TTATTGTC 28 (2) Information for SEQ ID NO: 23:

(i) Sequence characteristics:

(A) Length: 85 base pairs (B) Type: nucleic acid (C) Site type: single-stranded (D) Topology: linear (ii) Molecular type: DNA (genomic) (ix) Characteristics:

(A) Name / Key: Mixed Feature (B) Location: 1..85 (D) Other Information:, / Function = DNA Part with Sticky Ends / Note = Mutant Rat BetaA4 Peptide (i x) Characteristics:

(A) Name / Key: Mixed (B) Location: 1.

(D) Other Information (ix)

(A) Name / Key: Mixed (B) Location: 61 (D) Other Information Feature / Function = BglII Place Characteristic. 85 / function = overlapping in Fig. 24.

(xi) Sequence description: SEQ ID NO: 23:

AGATCTGATG GACGCTGAAT TTGGTCACGA CTCTGGTTTC GAAGTTAGAC 50

ACCAAAAGTT GGTTATCGGT GCTGAAGACG TTGGT ··········· · · · · ·

-58 (2) Information for SEQ ID NO: 24:

(i) Sequence characteristics:

(A) Length: 85 base pairs (B) Type: nucleic acid (C) Fiber type: single-stranded (D) Topology: linear (ii) Molecular type: DNA (genomic) (ix) Characteristics:

(A) Name / Key: Mixed Feature (B) Location: Complementary (1..85) (D) Other Information: / Function = Other DNA Part with Sticky Ends / Note = Overlapping Mutant Rat BetaA4 SEQ ID NO: 23 (ix) Distinctive features:

(A) Name / Key: Mixed Feature (B) Location: 79..85 (D) Other Information: / Function = EcoRI Location (ix) Features:

(A) Name / Key: Mixed Feature (B) Location: 1..25 (D) Other Information: / Function = Overlapping with 23.

(xi) Sequence description: SEQ ID NO: 24:

CCAATAGCCA CGACTTCTGC AACCAAGGTT GTTCCCACGA TAGTAACCAA 50

ACTACCAACC ACCACAACAG TAGCGAACTC TTAAG 85 (2)

Information on page 25.

for SEQ ID NO:

(i) Sequence characteristics:

-59 (A) Length: 85 base pairs (B) Type: nucleic acid (C) Fiber type: single-stranded (D) Topology: linear (ii) Molecular type: DNA (genomic) (ix) Characteristics:

(A) Name / Key: Mixed Feature (B) Location: 1..85 (D) Other Information: / Function = DNA with Sticky Ends / Note = Inverse Human BetaA4 Peptide Sense Using Yeast Codons (ix) Characteristics:

(A) Name / Key: Mixed Feature (B) Location: 1..6 (D) Other Information: / Function = BglII Location (ix) Features:

(A) Name / Key: Mixed Feature (B) Location: 61..85 (D) Other Information: / Feature = Overlapping 26.

(xi) Sequence description: SEQ ID NO: 25:

AGATCTGATG GCTATCGTTG TCGGTGGTGT TATGTTGGGT ATCATTGCTG 50

GTAAGAACTC TGGTGTTGAC GAAGCTTTCT TCGTT 85 (2) Information for SEQ ID NO: 26:

(i) Sequence characteristics:

(A) Length: 85 base pairs (B) Type: Nucleic acid · * · ·

-60 (C) Fiber type: single-stranded (D) Topology: linear (ii) Molecular type: DNA (genomic) (ix) Characteristics:

(A) Name / Key: Mixed Feature (B) Location: Complementary (1..85) (D) Other Information: / function = other part of DNA with sticky ends / j ele = ibetaA4 / note = overlapping with SEQ ID NO: 25, inverted human betaA4 (ix)

(A) Name / Key: Mixed Feature (B) Location: 1..25 (D) Other Information: / Function = Overlapping with 25.

SEQ ID NO: (ix)

(A) Name / Key: Mixed Feature (B) Location: 79..85 (D) Other Information: / Function = EcoRI Site (xi) Sequence Description: SEQ ID NO: 26:

ACCACAACTG CTTCGAAAGA AGCAAAACTT CGTTGTGGTG CAACTTATGC 50

CAAGACTGGT GTCTAAGCTT CGACTGACTC TTAAG 85 (2) Information for SEQ ID NO: 27:

(i) Sequence characteristics:

(A) Length: 42 amino acids (B) Type: amino acid (C) Fiber type: Single-stranded · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

-61 (D) Topology: linear (ii) Molecule type: peptide (ix) Characteristics:

(A) Name / Key: peptide

(B) Location: : 1. . . 42 (D) Individual Information: : / sign = betaA4 / A] = egyzés "Human betaA4 (xi) a sequence description: 27th SEQ ID NO: sequence: Asp Alá Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin 1 5 10 15 Leu Val Phe Phe Alá Glu Asp With Gly Ser Asn Lys Gly Down Ile 2 0 25 30 Gly Leu Met Val Gly Gly Val With Ile Ala 35 40

(2) Information for SEQ ID NO: 28: (1) SEQ ID NO:

(A) Length: 42 amino acids (B) Type: amino acid (C) Strain type: single-stranded (D) Topology: linear (ii) Molecular type: peptide (ix) Characteristics:

(A) Name / Key: peptide (B) Location: 1..42 (D) Other information: / sign = mrbetaA4 / Note mutant rat betaA4 (xi) Sequence description: SEQ ID NO: 28:

asp below Glu Phe Gly His asp Ser Gly Phe Glu With Arg His Gin Lys 1 5 10 15 Leu With Ile Gly below Glu asp With Gly Ser Asn Lys Gly below Ile Ile 20 25 30 Gly Leu Met With Gly Gly With With Ile below 35 40 (2) Inf nose siók a 22 ID number for sequence ι:

(i) Sequence characteristics:

(A) Length: 42 amino acids (B) Type: amino acid (C) Fiber type: single-stranded (D) Topology: linear (ii) Molecular type: peptide (ix) Characteristics:

(A) Name / Key: Peptide (B) Location: 1..42 (D) Other Information: / sign = ibetaA4 / note = inverted human betaA4

(xi ) THE s sequence : ia description: 29th SEQ ID NO: sequence: below Ile Val Val Gly Gly Val Met Leu Gly Ile Ile Alá Gly Lys Asn 1 5 10 15 Ser Gly Val Asp Glu Alá Phe Phe Val Leu Lys Gin His His Val Glu 20 25 30 Tyr Gly Ser Asp His Arg Phe Glu Under Asp 35 40 (2) Informations for SEQ ID NO: 30:

(i) Sequence characteristics:

(A) Length: 78 bp · · ·

-63 (Β) Type: nucleic acid (C) Fiber type: double-stranded (D) Topology: linear (ii) Molecular type: DNA (genomic) (ix) Characteristics:

(A) Name / Key: Mixed Feature (B) Location: 1..5 (D) Other Information: / Function = Excess EcoRI Site (ix) Features:

(A) Name / Key: Mixed Feature (B) Location: 6..74 (D) Other Information: / Product = Nuclear Localizing Signal from SW40 T Antigen (ix) Features:

(A) Name / Key: Mixed Feature (B) Location: 75..78 (D) Other Information: / Function = Overshot Spel Location (xi) Sequence Description: SEQ ID NO: 30:

AATTCATGGA CAAGGTCTTC AGAAACTCTT CCAGAACTCC ACCAAAGAAG 50

AAGAGAAAGG TTGAAGACCC AGCACTAG

Claims (6)

Patent claims A host organism transformed with at least one first and a second expression cassette, wherein the first expression cassette comprising one or more misfolded protein response elements (UPR) operably coupled to a reporter element; - the second expression cassette comprises a promoter operably linked to a signal sequence, a DNA encoding a protein whose folding is to be observed and a terminator; and wherein the first and second expression cassettes are not naturally occurring in the host, and wherein the protein to be monitored in the second expression cassette is selected from the group consisting of a prion, pS3, [beta] -amyloid peptide and functional derivatives thereof. The host organism of claim 1, which is capable of secreting protein in the white. The host organism of claim 1, which is a plant, insect, mammalian, fungal, or animal cell. The host organism of claim 1, which is a 4. The mushroom. 5. é 1 e s z t he s e j 6. A The host organism of claim 1, which is a The host organism of claim 1, which is a Saccharomyces cerevisiae. • · 7th The 1. According to claim 1 host , with that well analyzed, that the first expression cassette UPR BiP out It comes from. 8th The 1. According to claim 1 host , with that g embellished, that the first expression cassette 1. or SEQ ID NO its functional equivalent. 9. That 1. According to claim 1 host , with that well analyzed, that UPR is more than one has a copy is present first express tape cassette. 10th The 1. According to claim 1 host , with that well analyzed, that 2-5 copies of UPR are present the first expression cassette. 11th The 1. According to claim 1 host , with that characterized in that the reporter element a promoter containing operably linked to a DNA transcribed under the control of this promoter and a terminator. 12. The host of claim 11, wherein the promoter in the first expression cassette is strictly regulated. 13. claim of host with characterized by the promoter from the CYC1 and KAR2 promoters standing from group answer t. j u k k i. 14. claim of host with characterized by the reporter element one causes an effect deep easy to measure or transcription during translation obsession after. • · ··*· ·* · · • · · • · · · the • · · · · 15. According to claim 1 host with j e11eme fishing, that the reporter gene is one protein encode, the amount of which can be easily measured. 16th THE 15th According to claim 1 host with well analyzed, that reporter protein a β-galactosidase and the lucifeázból standing is selected from. 12th THE 11th According to claim 1 host with well analyzed, h o g y the thermometer is transcribed DNA in nature connecting thermometer, PHO5, α-factor and the SUC2 gene is selected from the group of terminators. 18th The 1. According to claim 1 host, with that j elephant, that promoter of the second expression cassette inducible promoter. 19. That ¹ · According to claim 1 host, with that j elephant, that the promoter of the second expression cassette a The protein whose folding is to be observed is selected from the group consisting of the promoter originally associated with the coding DNA CUP1, GAPDH, GAPFL, GAL (10), PYK, TPI, ADH, PRC1 and PGK. 20th 1. The host of claim 1, wherein the promoter of the second expression cassette is the GAPFL promoter or a functional derivative thereof. 21. A j 1. i g é η yo ο n t of host with characterized in that the second expression cassette signal sequence is SUG2, α-factor, KEX1, PHO5 and the glukoamrlaz s z i gna J s k veti , ci ay composed of groups there is selected. • ί ··· » 22. The host of claim 1, wherein the signal sequence of the second expression cassette is SUC2 or α. 23. The host of claim 1, wherein the protein to be observed in the second expression cassette has an unusual amount of hydrophobic moiety on its surface. The host of claim 1, wherein the protein to be observed in the second expression cassette is or contains a β-amyloid protein or a functional derivative thereof. The host of claim 1, wherein the terminator of the second expression cassette is selected from the group consisting of a terminator naturally associated with the DNA encoding the protein, an α-factor terminator and a SUC2 terminator. 26. The expression cassette of claim 1, comprising a promoter operably linked to a signal sequence, a DNA encoding a β-amyloid peptide or a functional derivative thereof, and a terminator. 27. A hybrid plasmid comprising the expression cassette of claim 26. 28. The hybrid plasmid of claim 27, characterized in that it is based on a two micron plasmid of S. cerevisiae. 29. A Method for Determining the Effect of a Compound on the Incidence of Misfolded Protein, by: ·· ···· -68, characterized in that the transformed host organism of claim 1 is cultured under appropriate conditions, the test compound is used and the amount of reporter gene activation is measured. 30. The method of claim 29, further comprising the steps of: - culturing the transformed host organism of claim 1 under suitable conditions, optionally inducing expression of the protein whose defective fold inhibition is to be monitored, optionally adding a compound that promotes defective fold or aggregation, adding the test compound and observing the transcription or translation of the reporter element or the transcription or translation product or the effects thereof. A compound identified by the method of claim 29. Use of a compound according to claim 32 in a method of treatment. Use of a compound according to claim 32 for inhibiting protein aggregation. Use of a compound according to claim 32, wherein For the treatment of Altzheimer's disease.