WO1995020652A1

WO1995020652A1 - Method of determining the activity of a regulatory factor, and use of the method

Info

Publication number: WO1995020652A1
Application number: PCT/EP1995/000297
Authority: WO
Inventors: Herbert Altmann; Wolfgang Wendler
Original assignee: Medigene Gmbh
Priority date: 1994-01-28
Filing date: 1995-01-27
Publication date: 1995-08-03
Also published as: DE19502584A1; DE19502584C2; AU1662595A

Abstract

The invention concerns a method of determining the activity of a regulatory factor, this activity being detected by means of a reporter system. To this end, gene arrays are provided for a first and second regulatory factor and for one or more reporter systems. The active first regulatory factor affects the activity or expression of the second regulatory factor which affects, in turn, the reporter system. Following addition of an inhibitory component, the activation of the reporter system is detected by the interaction between the first and second regulatory factors.

Description

Procedure for determining the activity of a regulatory factor and

Use this procedure

Description

The invention relates to a method for determining the activity of regulatory factors, this activity being detectable via the activity of a reporter system.

In living cells, metabolic functions are either performed continuously or only in certain development phases or based on external signals. In the case of signal transmission mediated by hormones, for example, the corresponding signals are transported via interactions between proteins from the outer cell membrane into the cell interior in order to be converted there, for example in the cell nucleus, into a request for division. Disrupting these interactions can unbalance a healthy cell and lead to growth disorders. For example, a degenerate cell grows into a cancer and spreads the deadly tumor over the entire body through the formation of metastases. Viruses such as HIV (Human Immunodeficiency Virus) or HPV (Human Papilloma Virus) intervene in the natural processes of the cell and misuse it to reproduce and then infect other cells.

It is obvious that such interactions can be used to deliberately intervene in the events of a cell, as long as the corresponding proteins are known and are accessible to a simple test. Such tests have been developed in recent years. Ultimately, they are based on the fact that a simple biochemical reaction, which can be detected by a color reaction and can be carried out thousands of times simultaneously, is made dependent on such an interaction between proteins. So-called in vivo assays allow these interactions, for example in yeast cells which are easy to grow and are also suitable for the formation of human proteins (Brent, R. et al., PCT publication WO 92/05286; Fields, S. et al., US 5,283,173).

The transcription of protein-coding genes is initiated by a multiprotein complex consisting of Pol II and a number of specific and general transcription factors. A large number of these factors have been isolated and characterized in recent years, giving first insights into the mechanisms of eukaryotic gene expression (Zawel et al., Curr. Opin. Cell. Biol. (1992) 4, pp. 488-495 ; Cortes et al., Mol. Cell. Biol. (1992) 12, pp. 413-421; Flores et al., J. Biol. Che. (1992) 267, pp. 2786-2793).

In addition to the basal transcription machinery, the DNA-binding transcription factors play a crucial role in stimulated gene expression.

The three characteristic features of transcription activators, such as proto-oncogenes or viral transcription factors and protein-protein interactions in signal chains or multiprotein complexes, which make them particularly suitable targets for investigations, are their high diversity, their specificity and their possible role in the development of Diseases. For example, more than 300 gene-specific transcription factors have been described to date and it is believed that approximately 3,000 such factors are encoded by the human genome. Similar to receptors on the cell surface, practically no factor and no protein-protein interaction are exactly the same. Each protein has a unique surface, making it a unique target. However, the DNA-binding and the stimulating activity need not be localized on a polypeptide chain (Weston et al., Cell (1989) 58, pp. 85-93). The division of these two properties allows, for example, the investigation of an interaction between two proteins X and Y, the DNA-binding part being located on a protein X and the transcription-active part on a protein Y (Fields et al., Nature (1989) 340 , Pp. 245-246). The specific inhibition of this interaction results in the loss of the stimulating activity of this element.

To date, methods are known for detecting inhibitors which inhibit the biological activity of oncoproteins (WO 92/05286). These methods are used to identify and classify inhibitory components by examining the ability of such a component to affect the expression of reporter genes. According to the aforementioned PCT publication, fusion genes are provided for carrying out this method, which code for fusion proteins. These fusion proteins bind to a binding site on the DNA, which DNA codes for the reporter genes, and thus mediate the expression of the reporter gene. If the expression of the reporter gene is examined, a decrease in the reporter gene expression is indicative of a component which inhibits the activity of the fusion proteins.

When using the methods mentioned, inhibitors are detected exclusively by a decrease in the expression of the reporter genes in question, ie it is a negative detection. The known detection system is disadvantageous in that it may reduce the sensitivity of a test system. If, for example, a reporter gene is in principle only weakly expressed, it is often not possible to make clear statements after adding inhibitors and thus further decreasing expression. These disadvantages are based on the fact that an added inhibitory component inhibits a transcription factor which influences the expression of reporter genes, ie it is a direct functional connection of inhibitor and reporter gene. In particular, a lack of expression after the addition of inhibitor is disadvantageous, since experiments are often based on selection of organisms, as briefly explained below. If, for example, the growth of cells is examined after adding inhibitors, the inhibited cells to be examined do not grow and must be verified by further experiments. It is therefore not possible to screen many different potential inhibitors. The lack of expression of reporter genes after inhibitor addition can also be due to the influence of other factors, for example the inhibitor on factors of the translation and replication mechanisms and the cell cycle. The place of action of the inhibitor can therefore often not be clearly defined, which results in a lack of specificity in the previous detection systems.

The object of the present invention is therefore to provide a method which does not have the aforementioned disadvantages, which enables fast, clear and specific statements about test results and which improves the sensitivity of test systems.

The invention solves this problem by the method specified in independent claim 1 and the use according to claim 63. Further preferred configurations, aspects and details of the method according to the invention are in the dependent claims 2 to 62 and 64 and 65, the drawings, tables and the preferred Embodiments set out. The present invention provides a significantly improved detection system for the activity of a regulatory factor. With the specified The method can also be used to detect an inhibitory component in the living organism by expression or increased expression of a reporter system, without the disadvantages of known methods occurring.

This significantly increases the sensitivity of the test system and enables the screening of inhibitor libraries of great complexity (over 10 - * - 'different molecules).

A new principle for screening for inhibitors and chemicals that modify corresponding activities is hereby used. The assay can also be used to identify previously unknown interactions.

The provision of at least one second regulatory factor is essential for the method according to the invention. The method described below is preferably carried out in host organisms, which should not, however, preclude carrying out corresponding methods outside an organism. Microorganisms, in particular bacteria, or eukaryotic cells, in particular yeasts, are used as host organisms. The bacterial strain Escherichia coli or the yeast strain Saccharomyces cerevisia are particularly preferred.

According to the present invention, as mentioned above, preferably in a host organism, at least one reporter system with at least one first gene arrangement which has at least one reporter gene is provided. The expression of the reporter genes serves as a detection system.

Furthermore, at least one first regulatory factor or the corresponding gene arrangement is provided. According to the present invention, the at least one first regulatory factor influences one or more second regulatory factors and not directly the activity of the ReporterSystems, which makes it possible for the first time to demonstrate the activity of a first regulatory factor with a positive signal.

The at least one second regulatory factor is encoded by at least one second gene arrangement. For reasons of simplification, it is not always expressly mentioned in the case of the components already mentioned and those mentioned below which are involved in the process that both one and more components such as genes, gene arrangements, regulatory factors, proteins etc. can be involved. However, it should be construed to include these possibilities. The activity of the second regulatory factor is preferably influenced by the first regulatory factor. The interaction of the second regulatory factor with components of the reporter system also influences the activity of the reporter system. Thus, according to the present invention, the activation of the reporter system is detected by adding an inhibitory component through the interaction of the first and second regulatory factors.

The first regulatory factor contains one or more regulatory components. If the first regulatory factor contains several regulatory components, the composite form in particular is the active form. The one or more regulatory components are often one or more regulatory proteins. The regulating proteins are preferably one or more transcription regulators, for example transcription factors. In a preferred embodiment, the transcription factor contains only one protein.

According to a further preferred embodiment of the method according to the invention, the

Transcription regulator at least two hybrid proteins, in particular two hybrid proteins which are encoded by a third gene arrangement provided.

The hybrid proteins are preferably a first hybrid protein, which is a fusion protein from a DNA binding domain and a first protein component, and a second hybrid protein, which is a fusion protein from an activation domain of the transcription regulator and a second protein component. The active transcription regulator is created by binding between the two protein components, which are also called targets.

According to the present method, the regulatory components of the first regulatory factor are not restricted to regulatory proteins. For example, the first regulatory factor can contain nucleic acids or other components.

The activity of the at least one first regulatory factor is influenced by inhibitory components. These inhibitory components are preferably natural substances such as peptides, nucleic acids and carbohydrates or low molecular weight substances or other chemical substances or also components of the first regulatory factor changed by mutagenesis.

In a further preferred embodiment, a fourth gene arrangement for the expression of peptides is provided. These peptides in particular have an inhibitory activity. Peptide libraries synthesized in vivo can thus be used to inhibit regulatory factors. In further preferred configurations, any combinations of the inhibitors mentioned are added. The action of the inhibitory components on the activity of the first regulatory factor by action on the interaction between at least two regulatory components contained in the first regulatory factor is particularly important. This action is preferably an inhibition of the interaction of the regulatory components. The activity can also be regulated on the basis of several components, for example in a multiprotein complex. This complex is active as long as single or multiple building blocks form this complex. Only the inhibition of one or more of these components destroys the activity of the entire complex. Activities which deviate from what has been described so far and which in some way activate the expression of the second regulatory factor are also suitable as the first regulatory factor. The inhibition of the first regulatory factor can affect both the activity of the transcription regulator and / or the generation of the transcription regulator. In particular, the interaction between two regulatory proteins of the first regulatory factor is inhibited. If one of the interactions mentioned is inhibited, there is an inactive or reduced first regulatory factor in its activity. As a result, the interaction of the first regulatory factor with the second regulatory factor is inhibited or reduced. In a further preferred embodiment, the interaction between the first regulatory factor and the second regulatory factor is influenced. This action is also particularly preferably an inhibition. In a particularly advantageous embodiment, the inhibitor influences the interaction of the first regulatory factor with gene segments of the second gene arrangement. In a further embodiment of the method according to the invention, the at least one first regulatory factor, which preferably contains one or more proteins, modifies the at least one second regulatory factor, for example via kinization, dephosphorylation, cleavage, refolding or change of conformation.

If no inhibitor is added, the first regulatory factor is in particular in the active form and acts on the activity of the second regulatory factor. It is further preferred that the first regulatory factor interacts with DNA segments of the second gene arrangement coding for the second regulatory factor and thus influences the expression of the second regulatory factor.

The addition of an inhibitory component influences, for example, the interaction between at least two components which together form the first regulatory factor. In a further embodiment, the addition of the inhibitory component acts on the activity of the first regulatory factor, regardless of whether it contains one or more regulatory components. The action on the activity relates, for example, to the transcription-activating or binding activity of the first regulatory factor or the interaction of the first and second regulatory factors.

In a further embodiment, the addition of an inhibitory component influences both the interaction mentioned and the activity mentioned. The action is preferably an inhibition of the interaction and / or the activity.

By adding the inhibitory component, the interaction between the first regulatory factor and a DNA section of the second gene arrangement which codes the second regulatory factor, which is in particular an inhibition, is preferably influenced. The action or inhibition of the above-mentioned interactions by inhibitory components leads to an action on the gene expression of the second Gene arrangement, in particular to inhibit gene expression of the second gene arrangement. The addition of the inhibitory component particularly preferably leads to an inhibition of the expression of the second gene arrangement by inhibiting the activity of the regulating protein.

According to a further preferred embodiment of the method according to the invention, the interaction between at least two components is influenced by the addition of an inhibitory component, one of these components being a regulatory component or containing a regulatory component.

It is advantageous if this regulatory component is a protein component or contains at least one protein component. Fusion proteins are preferred for the protein components.

According to a particularly preferred embodiment, the regulatory component is an inhibitory component.

The at least one second component is, for example, a protein component or contains a protein component. It has proven to be advantageous that this protein component is a fusion protein.

The second protein components are, in particular, protein components which have anchoring functions. The mutually interacting protein components are preferably anchored in the cytoplasm. Anchoring the interacting proteins via the anchoring function of the second protein component in the membrane has also proven to be advantageous. According to a further advantageous embodiment, the addition of an inhibitory component inhibits the interaction between the at least two components and releases the at least one first component which contains the inhibitory section. This released first component interacts with the transcription activating factor of the gene arrangement for the second regulatory factor. The transcription-activating factor is particularly preferably inhibited by the released inhibitory factor, as a result of which the expression of the second gene arrangement is inhibited.

In the embodiments mentioned so far and also in the following, the regulatory or interacting components contain sections which cause the regulatory or interacting functions. The corresponding protein segments can contain one or more segments or domains. These sections or domains can be located in different regions of a protein or on different proteins.

The at least two protein components are preferably encoded by a fifth gene arrangement.

It has proven to be particularly advantageous that the at least one first protein component contains a section which interacts with the at least one second protein component, a section which interacts with a transcription factor and an inhibitory section, only after inhibiting the interaction of the bond between the at least two protein components, the inhibitory second protein component inhibits the expression of the second regulatory factor.

According to a further preferred embodiment, the at least one first regulatory component is one Transcription regulator or transcription factor of the second regulatory factor, the activity of which is reduced or inactivated after inhibiting the interaction with the at least one second protein component, as a result of which the activity of the second regulatory factor is inhibited or reduced.

The second regulatory factors are in particular proteins, for example, depending on the experimental arrangement, at least one repressor or at least one recombinase. The active second regulatory factors act on the activity of the reporter system via an interaction with components of the reporter system. The second regulatory factor preferably binds to DNA sections of the reporter system.

A repressor encoded by the second gene arrangement is an example of a second regulatory factor. This repressor influences the expression of at least one reporter gene by preferably binding to components of the reporter system. According to a preferred embodiment of the method according to the invention, the binding of the repressor to components of the reporter system is regulated by further agents. For example, the antibiotic tetracycline induces a prokaryotic repressor to detach from DNA. An active repressor inhibits the expression of at least one reporter gene. If, on the other hand, the expression of the repressor gene is inhibited, for example by inhibiting the interaction of the hybrid proteins of the transcription regulator, the expression of at least one reporter gene takes place.

A recombinase encoded by the second gene arrangement is another example of a second regulatory factor. In a corresponding test arrangement, the reporter systems contain recombination elements. There is an active one first regulatory factor before, eliminates or inverts the recombinase via recombination processes at least one reporter gene. Here, the recombinase interacts with the specific recombination elements flanking the reporter gene or the reporter genes and eliminates or inverts the reporter gene which is flanked by the recombination elements. The reporter gene is not expressed either after elimination or after inverting. If no inhibitor has been added to the experiment, there is an active first regulatory factor that preferably activates the genes coding for a second regulatory factor, in particular via interaction with DNA segments. The second active factor, such as a repressor or a recombinase, inhibits the expression of the reporter genes.

In a preferred embodiment of the method according to the invention, the expression of at least one recombinase is controlled by influencing the interaction of at least two regulatory components of the transcription regulator, the regulatory components being in particular two hybrid proteins, whereby a change in the expression of at least one reporter gene takes place. By inhibiting the interaction of the hybrid proteins of the transcription regulator (or other regulatory factors of the transcription regulator), the expression of the recombinase is particularly inhibited and the at least one reporter gene is expressed.

An embodiment is particularly preferred in which the expression of at least one repressor gene is controlled by the action on the interaction of the at least two protein components, as a result of which the expression of the at least one reporter gene is changed. In particular, by inhibiting the interaction of the protein components, the expression of the repressor gene is inhibited and the at least one reporter gene is expressed.

According to a further preferred embodiment of the invention, the action on the interaction of the at least two protein components controls the expression of at least one recombinase, as a result of which the expression of at least one reporter gene is changed.

The expression of the recombinase is particularly preferably inhibited by inhibiting the interaction of the protein components and the at least one reporter gene is expressed.

The tests are based on the fact that at least one gene product of the at least one reporter gene can be detected under the given test conditions.

According to preferred embodiments, one gene product of one reporter gene or several gene products of several reporter genes are expressed. In a further embodiment, one or more reporter genes are expressed depending on the varying test conditions.

The detection of the gene product or the gene products then takes place, for example, via one or more changes in the phenotype of host cells. In a particularly preferred embodiment, the gene product of the reporter gene enables cell growth of the host cells in deficient medium. The gene product, for example of the reporter gene Leu2, enables cell growth in leucine-deficient medium. In a further advantageous modification of the method according to the invention, yeast strains with chromosomal mutations are used as host cells, which lead, for example, to leucine deficits in the metabolism of the amino acid leucine. The Chromosomal mutations can also lead to deficiencies in the metabolism of the amino acids tryptophan and histidine. If necessary, the use of protease-deficient yeasts also makes sense.

It is also preferred to provide gene arrays that code for one or more gene products, which gene products can convert substrates in a measurable color reaction, e.g. the LacZ reporter system. The gene product of this reporter gene, the ß-galactosidase, reacts with various substrates in a visible color reaction.

The gene arrangements mentioned for the present method can be arranged on different vectors or on the same vector. Plasmids in particular are used as vectors. In a further preferred embodiment, one or more vectors with one or more gene arrangements, or one or more gene arrangements, are integrated into the host genome.

According to the present invention, the use of one or more inhibitory components initially influences the activity of the first regulatory factor, which in turn affects the activity of the second regulatory factor.

The second regulatory factor ultimately affects the expression of the reporter gene. According to the present invention, the activity of the first regulatory factor is preferably inhibited by the addition of one or more inhibitory components; this also causes the second regulatory factor to be inhibited, the second regulatory factor itself being inhibited according to a preferred embodiment, or expression in a further preferred embodiment of the second regulatory factor is inhibited. Since in no case active second regulatory factor is present, the reporter gene or the reporter genes are expressed. It is only when the first regulatory factor is inhibited that reporter genes are expressed. The respective phenotype depends on whether the second regulatory factor is formed or not. Another embodiment of the present invention is to be particularly emphasized, in which two gene arrangements for two second regulatory factors, in particular gene arrangements for a repressor and a recombinase, are provided in a host organism, in addition to the other gene arrangements necessary for carrying out the experiment.

Depending on the test condition, one of the two second regulatory factors, i.e. especially repressor or recombinase, or both are expressed simultaneously. This results in an increased specificity of the method.

The inhibition of the activity of the transcription factor or the transcription regulator or its generation ultimately leads to the activation of the reporter gene or the reporter genes.

According to the method of the present invention, it is possible to detect highly specific inhibitors and to increase the selectivity and specificity of the detection of inhibitors. With the present method, for example, in growth experiments, the cells to which the inhibitor has acted can be selectively detected, while the "uninhibited" cells do not grow, ie there are no problems such as overgrowth of the cells of interest. This significantly increases the sensitivity of the test system and enables the screening of inhibitor libraries of great complexity (over 10- ^* - 'different molecules). It is thus the first time with the method according to the invention It is possible to carry out identification of cells after the addition of inhibitor, in particular also due to growth. This proves to be very advantageous if, for example, there is a very unfavorable ratio of cells on which the inhibitor acts (usually a very small proportion) to cells on which the inhibitor does not act. It is also possible. To make statements regarding the specific site of action of the inhibitor, since it acts specifically on, for example, regulatory factors and the reporter genes are only expressed after the factor has been inhibited. The method enables the investigation of signal chains and other regulatory mechanisms and offers the prerequisites for specific drug development. The method is used to find lead structures that are preferably used for the development of therapeutic agents. Furthermore, the method is preferably used to determine inhibiting substances that can be used, for example, as lead structures. These inhibiting substances are, for example, peptides, natural products and synthetic chemicals. In a particularly preferred embodiment, peptide libraries synthesized in vivo are provided to inhibit the first regulatory factor.

In the following, preferred embodiments of the method according to the present invention are presented, the execution being explained in each case taking into account an active or inactive first regulatory factor. The starting point for the execution of proteins as regulatory factors.

Serve as reporter gene e.g. the Leu2 gene, which enables growth on leucine-deficient medium, and the LacZ gene, whose gene product, the β-galactosidase, converts various substrates in a visible color reaction (X-Gal (5-bromo-4-chloro-3- indoxyl-β-D-galactopyranoside)

Blue color, ONPG (o-nitro-phenyl-galactopyranoside) results Yellowing). Analogously to Leu2, other genes that encode enzymes from biosynthesis, such as the URA3 gene, can also be used, since its expression can complement deficient yeast strains and thus enable growth on uracil-deficient media (Sherman et al., Laboratory Course Manual for Methods, In: Yeast Genetics, (1986)). The activity of luciferase or chloramphenicol acetyltransferase can also be easily and quickly detected in enzymatic reactions (Ibelgaufts, Genotechnologie from A to Z (1990) VCH-Verlag (Weinheim)).

The invention is explained below with reference to the accompanying drawings.

Fig. La and lb show schematically shown

Gene arrangements for a repressor and for the reporter proteins, the

Interaction of an active (Fig. La) and an inactive (Fig. Lb) transcription factor with the DNA is shown.

2a and 2b show schematically shown

Gene arrangements for a repressor and for the reporter proteins, the

Interaction of an active (FIG. 2a) and an inactive (FIG. 2b) transcription regulator with the DNA is shown.

3a and 3b show schematically shown

Gene arrangements for a recombinase and for reporter proteins, the interaction of an active (FIG. 3a) and an inactive (FIG. 3b) transcription factor with the DNA being shown. 4a and 4b show schematically shown

Gene arrangements for a recombinase and for reporter proteins, the

Interaction of an active (FIG. 4a) and an inactive (FIG. 4b) transcription regulator with the DNA is shown.

5a and 5b show schematically shown

Gene arrangements for a repressor and for the reporter proteins, the

Interaction of an active (FIG. 5a) and an inactive (FIG. 5b) transcription regulator with the DNA is shown.

6a and 6b show schematically shown

Gene arrangements for a recombinase and for reporter proteins, the interaction of an active (FIG. 6a) and an inactive (FIG. 6b) transcription regulator with the DNA being shown.

7a and 7b show schematically shown

Gene arrangements for a repressor and for the reporter proteins, the interaction of an active (FIG. 7a) and an inactive (FIG. 7b) transcriptional regulator with the DNA being shown.

8a and 8b show schematically represented gene arrangements for a recombinase and for reporter proteins, the interaction of an active (FIG. 8a) and an inactive (FIG. 8b) transcriptional regulator with the DNA being shown. 9a and 9b show schematic representations

Gene arrangements for a LexA repressor

GST, the interaction of an active (Fig. 9a) and an inactive (Fig. 9b) first regulatory factor

ADl-Tet is shown with the DNA.

10a and 10b show schematically shown

Gene arrangements for a second regulatory factor Tet-GST, the

Interaction of an active (Fig. 10a) and an inactive (Fig. 10b) first regulatory factor LexA-CTF7 with the DNA is shown.

11a and 11b show schematically shown

Gene arrangements for a second regulatory factor Tet-GST, the interaction of an active (FIG. 11a) and an inactive (FIG. 11b) first regulatory factor LexA-CTF2-ADl-TIM with the DNA being shown.

The targets or targets mentioned in FIGS. 1 and 3 or transcription-stimulating targets are regulatory factors, in particular transcription factors for the expression of the second regulatory factors, on which the inhibitor acts. The regulatory factors in the examples shown in the figures are a repressor or a recombinase.

2 and 4 are the interacting regulatory components of the regulatory factor, target I being the regulatory component with the binding activity and Target II is the regulatory component with the transcription activating activity.

The targets mentioned in FIGS. 5 and 6 are those interacting with one another

Components of the first regulatory factor, where Target

I the regulatory component with both the DNA-binding and the transcription-activating activity and target

II is the component with which Target I interacts and only with undisturbed interaction in the active one

Form is present.

The targets mentioned in FIGS. 7 and 8 are interacting components, target II being the regulatory component with both an inhibitory activity and a binding activity, and target III being an anchored component.

The binding activity of Target II relates to the interaction of Target II, which has been released by Target III, with the X component of Target I. The X component of Target I, in turn, is an activating transcription factor of the second regulatory factor.

The term activity denotes in particular protein segments which bring about the respective activity or the relevant function. One or more domains can be affected. The domains or protein segments can be located in different areas of a protein or on different proteins.

Accordingly, the binding sites of these factors are referred to as target binding sites.

In Examples 1 to 11, the procedure was as follows: Yeast plasmids

Both commercially available plasmids (eg pYES2 from Invitrogen, the Netherlands) and other constructs (Altmann, H. Dissertation (1994), Altmann H. et al., Proc. Natl. Acad.) Are used to express the various genes in the underlying assay Se., USA (1994) 91, pp. 3901-3905). Normally, these plasmids have a replication origin for the replication of their DNA in yeast (e.g. "2μm-ori") and one for replication in bacteria (e.g. "colEl-Ori"). Furthermore, marker genes are used to select these plasmids in the two organisms (e.g. URA3, HIS3, TRPl or LEU2 in yeast and Ampr or Tetr in E. coli) (Sherman et al., Laboratory Course Manual for Methods, In: Yeast Genetics, ( 1986); Ibelgaufts, genetic engineering from A to Z (1990) VCH-Verlag (Weinheim)). Examples of yeast vectors are described (Winnacker et al., From Genes to Clones (1987) VCH-Verlag (Weinheim); The Molecular and Cellular Biology of the Yeast Saccharomyces (1991) Vol. 1 and 2, Cold Spring Harbor Laboratory Press).

Genes can be expressed via constitutive (e.g. ADHI promoter), inducible (e.g. Gall promoter) or specially designed target-dependent promoters (NFI, Tet, LexA).

Instead of being cloned into plasmids, the individual elements of the assay can also be integrated into the genome of the yeast strain used (Sherman et al., Laboratory Course Manual for Methods, In: Yeast Genetics, (1986)). Elements located in the yeast in this way do not require their own origin of replication or selection markers.

The following elements are used in the test system: Promoters:

ADHI promoter: This constitutively expressing promoter is functionally isolated from the plasmid pAAH5 via the restriction with the enzymes BamHI and HindIII (Ammerer et al., Methods in Enzymology (1983) Academic Press, pp. 192-201).

Gall promoter: This promoter, which is repressible on media containing glucose and inducible on media containing galactose, is isolated from the vector pYES2 from Invitrogen (Netherlands) with the aid of the restriction enzyme Spei.

Inactive Gall / GallO promoter: This promoter, which was originally inducible via galactose, is deleted at the sequence level in such a way that it only has basic activity in yeast (Yocum et al., Mol. Cell. Biol. (1984) 4, pp. 1985-1998 ; West et al., Mol. Cell. Biol. (1984) 4, pp. 2467-2478). With the help of the restriction enzymes BamHI, EcoRI and HindiII, this promoter element is isolated from the vector pLRlΔl thus constructed and used for further purposes.

NFI-dependent promoter: The by Meisterernst et al. (Nucl. Acid Res. (1988) 236, pp. 27-32) found consensus sequence as a DNA binding site for members of the NFI family (nuclear factor I) is synthesized as an oligonucleotide and used for the construction of NFI-dependent promoters (Altmann et al., Proc. Natl. Acad. Sci. USA (1994) 91, pp. 3901-3905).

Sequence for 6 NFI binding sites constructed from two oligonucleotides: 1 . Oligo:

5'- TCG AGT TTT TGG CAC TGT GCC AAT TCT TTT TGG CAC TGT GCC AAT TCT 3'- CA AAA ACC GTG ACA CGG TTA AGA AAA ACC GTG ACA CGG TTA AGA TTT TGG CAC TGT GCC AAT TC - 3 'AAA ACC GTG ACA CGG TTA AG - 5 '

2. Oligo:

5'- TTT TTG GCA CTG TGC CAA TTC TTT TTG GCA CTG TGC CAA TTC TTT TTG 3'- AAA AAC CGT GAC ACG GTT AAG AAA AAC CGT GAC ACG GTT AAG AAA AAC GCA CTG TGC CAA TTC - 3 '

CGT GAC ACG GTT AAG AGC T - 5 '

Tet-dependent promoters: The methods described by Hillen et al. (Nature (1982) 297, pp. 700-702) found consensus sequence as a DNA binding site for the Tet repressor is synthesized as an oligonucleotide and used for the construction of Tet-dependent promoters.

Sequence for the Tet operator binding site O-j / 02:

5'- TCG ATC TCT ATC ACT GAT AGG GAG TGG TAA AAT AAC TCT ATC AAT GAT 3'- AG AGA TAG TGA CTA TCC CTC ACC ATT TTA TTG AGA TAG TTA CTA AGA - 3 'TCT CAG A- 5'

LexA-dependent promoters: The DNA consensus sequence as the binding site for the bacterial repressor LexA was synthesized as an oligonucleotide and used for the construction of LexA-dependent promoters (Altmann et al., Proc. Natl. Acad. Sei. USA (1994) 91, Pp. 3901-3905; Wendler et al., Nuc. Acid Res. (1994) 22, pp. 2601-2603; Brent et al., Cell (1985) 43, pp. 729-736). In order to demonstrate the repressing activity of LexA fusions on the galactose-induced gall promoter, this promoter is extracted from the plasmid JK101 (Golemis et al., Mol. Cell. Biol. (1992) 12, pp. 3006-3014) isolated and used for further cloning.

loxP-dependent promoters: The Hoess et al. (Proc. Natl. Acad. Sci. USA (1982) 79, pp. 3398-3402) described loxP elements which make it possible for the recombinase Cre of Coliphagen P1 to delete or invert sequences lying between two such elements are synthesized as oligonucleotides and used for the construction of Cre-dependent promoters.

Sequence for the loxP recombinase element:

5'- GAG ATC ATA TTC AAT AAC CCT TAA TAT AAC TTC GTA TAA TGT ATG CTA

3'- CTC TAG TAT AAG TTA TTG GGA ATT ATA TTG AAG CAT ATT ACA TAC GAT

TAC GAA GTT ATT AGG TCG - 3 '

ATG CTT CAA TAA TCC AGC AGC T - 5 '

Reporter genes.

LacZ gene: The LacZ gene is isolated via the restriction enzymes BamHI and HindIII from the plasmid pMC1871 (Casadaban et al., J. Meth. In Enzy. 100, (1983) 100, pp. 293-308) and for the others Cloning used.

Leu2 gene: The Leu2 gene is isolated from the plasmid pAAH5 (Ammerer, Methods in Enzymology (1983), pp. 192-201, Academic Press) by means of a PCR reaction and used for the further cloning.

Sequence primers used:

Leul: 5'- CGC GGA TCC ATG TCT GCC CCT AAG - 3 'Leulrev: 5'- GCT CTA GAT CTT TTT AAG CAA GGA TTT TC - 3' Tet gene: The Tet gene is isolated using the restriction enzymes Xbal and BstEII from the plasmid pWH1950, a derivative of the plasmid pRT240 (Wissmann et al., Genetics (1991) 128, pp. 225-232) and used for the further cloning .

Crel gene: The Crel gene is isolated by means of a PCR reaction from Coliphagen Pl (Hoess et al., J. Mol. Biol. (1985) 181, pp. 351-363) and used for the further cloning.

Sequence primers used:

Crel: 5'- GGG GTA CCT ATG TCC AAT TTA CTG AC - 3 'Crelrev: 5'- GGG GTA CCG CGG CCG CCT AAT CGC CAT CTT CC - 3'

c) target genes

NFI genes: The various NFI genes are cloned and used for the further cloning (Meisterernst et al., Nuc. Acid Res. (1988) 236, pp. 27-32; Altmann et al., Proc. Natl. Acad. Sci. (1994) 91, pp. 3901-3905).

LexA gene: The LexA gene is generated with the aid of the restriction enzymes HindiII and EcoRI from the plasmid pEG202, a derivative of the vector LexA202 with an additional polylinker sequence behind the LexA gene (Rüden et al., Nature (1991) 350, p. 250 -252) isolated and used for further cloning.

NFkB gene: The coding sequence for the transcription-active domain TA ^ (amino acid 521-551) of the NFkB protein is generated from the plasmid pLexTA- ^ using the restriction enzyme EcoRI (Schmitz et al., J. Biol. Chem. (1994) 269 , Pp. 25613-25620) isolated and used for the further cloning. TIM gene: The coding sequence for the C-terminal part of the TIM protein is with CTF2, a member of the NFI family, in the so-called "interaction trap" (Current Protocols in Molecular Biology, 1994; Altmann (1994) dissertation) isolated.

GST gene: The coding DNA sequence for the 26 kDa domain from the GST protein (glutathione-S-transferase) is isolated from the commercially available plasmid pGEX 3X from Pharmacia (Sweden) using restriction enzymes and used for further cloning.

ADl-Tet: The fusion gene ADl-Tet is composed of the acidic activation domain ADl, isolated from the plasmid pJG4-5 (Gyuris et al., Cell (1983) 75, pp. 791-803) with the aid of the restriction enzymes Hindlll and EcoRI, and the coding sequence for the Tet repressor (see above).

Inhibitor expression:

TrxA gene: The TrxA gene is isolated from the plasmid pCJF7 (Lim et al., J. Bact. (1985) 163, pp. 31-36) by means of a PCR reaction and used for the further cloning.

Sequence primers used:

Trxl: 5'- GGA ATT CCC CGG GAT GAG CGA TAA AAT TAT TC - 3 'Trxlrev: 5'- CGG GAT CCC TCG AGT CAG CTA ATT ACC CGG GTA CCA CTT G -3'

"Random oligo pool": To construct a "random oligo pool", a single-strand oligonucleotide pool of the following composition is synthesized: Sequence:

5'- TAG CCG ATT TAG GTG ACA CTA TAG AGA GCT CTT CCG TCT GGC TAG CNN BNN BNN BNN BNN BNN BNN BNN BNN BNN BNN BAG CGC TGG TCC GAA GAG CTC TCC CTA TAG TGA GTC GTA TTA ..- 3 '

N means that at this point all 4 possible bases (A, G, C and T) were added during the synthesis (IUPAC nomenclature).

B means that the three bases G, C and T were added during the synthesis (IUPAC nomenclature).

Double strands are produced from the single strands by means of Klenow polymerase.

These were then cloned into the RsrII site of the TrxA gene after incubation with the restriction enzyme SapI.

Experimental approach

The cultivation and transformation of bacteria, yeast and higher eukaryotic cells takes place according to standard conditions (Ausubel et al., Current Protocols in Molecular Biology (1987), John Wiley & Sons (New York); Miller, Experiments in Molecular Genetics (1972), Cold Spring Harbor, NY; Sambrook et al., Molecular Cloning (1989), Cold Spring Harbor Laboratory Press; Lindl et al., (1987); Altmann et al., Proc. Natl. Acad. Sei. (1994) 91, S 3901-3905).

The bacterial strains JK101 from Stratagene (Germany) and ToplO from Invitrogen (Germany), as well as the yeast strains INVScl and IΝVSc2 from ITC (Germany) were used for the experiments. The determination of the β-galactosidase activity, the LacZ gene product, is carried out in so-called β-galactosidase assays (Altmann et al., Proc. Natl. Acad. Sei. (1994) 91, pp. 3901-3905).

To determine the CAT enzyme activity in protein extracts according to the immunoassay principle, the "CAT ELISA" test commercially available from Boehringer (Germany) is used, for example.

example 1

This example represents the configuration shown in FIG. 1.

1. A gene arrangement is provided which codes for a transcription factor. This transcription factor binds to DNA regions of a DNA (target binding site) which codes for a second regulatory factor, in this test arrangement for a repressor.

2. Furthermore, a repressor gene arrangement is provided with a binding site on the DNA (target binding site) for the transcription factor mentioned under 1.

3. A reporter gene arrangement with the reporter genes Leu2 and / or LacZ is also provided, the structure of both reporter genes in the present method having the same regulable promoter. This promoter consists of so-called UAS elements (upstream activation sequence), to which an endogenous activator (further

Transcription factor TF) binds from one or several repressor recognition sequences to which a repressor can bind, and a TATA box for basal transcription.

a. If no inhibitor is added to the experimental set-up, there is an active transcription factor that positively regulates the expression of a repressor protein. The active repressor binds to the repressor recognition sequence or repressor binding site mentioned above and represses the expression of the reporter gene LacZ and / or Leu2, i.e. there is no growth of the host organisms, there is no evidence of a color reaction.

b. After adding an inhibitor, the activity of the first regulatory factor, here the transcription factor, is inhibited. This is due to an inhibition of the interaction of the transcription factor with the corresponding binding site on the DNA or an inhibition of the activation domain. There is no expression of the repression morning. Thus, no repressor is available and the LacZ and / or Leu2 reporter gene downstream of the promoter is replaced by an inducible endogenous one

Highly expressed transcription factor. As a result, the yeasts grow in leucine-deficient media, and after growth in medium containing X-Gal, a color change (blue) occurs.

According to the present invention, the addition of an inhibitor leads to expression of the reporter gene (s) by inhibiting the second regulatory factor, here repressor. Example 2

This example represents the configuration shown in FIG. 2.

1. Gen arrangements are provided which code for two interacting hybrid proteins of the transcription regulator and contain a UAS sequence and a TATA box, an arrangement coding for a hybrid protein which is a fusion protein from a binding domain and a first protein component, and encoded a further arrangement for a hybrid protein, which is a fusion protein from an activation domain of the transcription regulator and a second protein component (FIG. 2a).

2. Furthermore, a repressor gene arrangement with one or more binding sites on the DNA (target binding site) is provided for the transcription regulator.

3. A reporter gene arrangement as described under Example 1, point 3 is also provided.

a. As described above, the transcription regulator is composed of two fusion proteins, an active transcription regulator being present only when the protein-protein interaction of the two fusion proteins is undisturbed (see FIG. 2a). If there is no inhibitor, an undisturbed protein-protein interaction takes place and an active transcription regulator is present. This active transcription regulator binds to its corresponding binding site on the DNA (target binding site), which is responsible for the

Repressor gene encodes, and causes expression of the repression morning. The repressor binds to the repressor binding site in the promoter region of the reporter genes and represses expression of the reporter genes. Since Leu2 and / or LacZ are not expressed, no growth is found

Leucine-deficient medium takes place, there is still a blue color after growth on X-Gal-containing medium.

If an inhibitor is added to the experimental set-up, e.g. the inhibitors mentioned above, for example peptides, nucleic acids, carbohydrates or other chemical substances, the protein-protein interaction of the fusion proteins of the

Transcription regulator inhibited. There is no active transcriptional regulator or one with reduced activity

Transcription regulator before, since the activation domain of the transcription regulator and the DNA binding domain are located on different fusion proteins and consequently, if the interaction of the hybrid proteins is inhibited, these domains cannot interact with one another or to a lesser extent.

The DNA-binding domain of the one fusion protein of the transcriptional regulator can, depending on the test conditions and the inhibitor added, bind or not bind to the relevant DNA section. Due to the inhibited protein-protein interaction, there is no active transcriptional regulator in any case. Due to the inactive transcription regulator, there is no expression of the repressive morning. Since there is no repressor, the reporter genes Leu2 and / or LacZ are expressed after binding a corresponding further transcription factor. There is now growth of the host cells on leucine-deficient medium

Color change (blue color) after growth on X-gal-containing medium is detectable. According to the present invention, the addition of an inhibitor results in inhibition of the second regulatory factor (repressor)

Expression of the reporter genes.

Further preferred embodiments result from modifications of the embodiments described in Examples 1 and 2.

Example 3

This example relates to the configuration shown in FIG. 3.

1. A gene arrangement which codes for a transcription factor is provided. This transcription factor binds to regions of a DNA (target binding site) which codes for a recombinase.

2. Furthermore, a gene arrangement is provided which codes for the sequence-specific recombinase of Coliphagen P1.

3. A reporter gene arrangement with the reporter genes Leu2 and / or LacZ is provided, the structure of both reporter genes in the present method having the same regulatable promoter. This

Promoter contains UAS elements to which preference is given endogenous activator binds, and a TATA box. The reporter genes have flanking lox P sequences as recombination elements.

a. As already described under Example 1, unless an inhibitor is added, there is an active transcription factor. This

Transcription factor positively regulates the expression of the recombinase Cre. The recombinase interacts with the lox P sequences that the

Reporter genes LacZ / Leu2 flank and eliminates or inverts the reporter genes mentioned in a recombination process.

Accordingly, no functional parts

Reporter genes are expressed, there is no growth of the host organisms, and there is also no detectable color reaction after growth on medium containing X-Gal.

b. After adding an inhibitor, the activity of the transcription factor is inhibited, this being due to an inhibition of the interaction of the transcription factor with the corresponding binding site on the DNA or to an inhibition of the

Activation domain. As a result, there is no expression of the recombinase and the reporter genes LacZ and / or Leu2 downstream of the promoter are strongly expressed by an inducible, preferably endogenous, transcription factor. The host organisms, in this experimental arrangement it is preferably yeasts, now grow on leucine-deficient media and when they grow in X-gal-containing medium, a color change (blue) occurs. The addition of an inhibitor in turn leads to expression of reporter genes.

Example 4

This example relates to the configuration shown in FIG. 4.

1. There are gene arrangements as described in Example 2, provided for interacting

Coding hybrid proteins that form an active transcriptional regulator via a protein-protein interaction.

2. Furthermore, a gene arrangement is provided which codes for the sequence-specific recombinase Cre of Coliphagen P1. This recombinase corresponds to the second regulatory factor.

3. A reporter gene arrangement with the reporter genes Leu2 and / or LacZ is also provided, the structure of both reporter genes in the present method having the same regulable promoter. This promoter contains UAS elements, to which an endogenous activator preferably binds, and a TATA box. The reporter genes have flanking lox P sequences as recombination elements.

a. As also described under Example 2, if there is no inhibitor, an undisturbed one is found

Protein-protein interaction takes place between the fusion proteins forming the transcription factor, with which there is an active transcription factor. The active transcription factor binds to a binding site on the DNA, this DNA being the one for the Cre recombinase contains encoding cre gene. After the transcription factor has been bound, the Cre recombinase is expressed. This recombinase eliminates or inverts the reporter gene or genes via recombination processes, with the lox P-

Sequences that flank the reporter genes interact. No functional reporter genes are expressed either after elimination or after inversion. Accordingly, there is no growth on leucine-deficient medium without the addition of an inhibitor, and likewise no blue color when growing on medium containing X-Gal.

After adding an inhibitor to the test batch, the protein-protein interaction of the fusion proteins of the transcription regulator is inhibited. There is therefore no active transcription regulator. The DNA binding domain of a fusion protein of the

Depending on the experimental conditions and the inhibitor added, the transcription regulator binds to the relevant DNA segment or not. Because of the inhibited protein-protein interaction there is no active one

Transcription regulator. Due to the lack of an active transcription regulator, no transcription regulator binds to the binding site on the DNA which codes for the Cre recombinase. The Cre recombinase is therefore not expressed.

Accordingly, the reporter genes Leu2 and / or LacZ are neither eliminated nor inverted. A lack of recombinase thus leads to expression of the reporter genes, which enables growth of the cells in leucine-deficient medium is or a blue color occurs in X-Gal-containing medium.

According to the present invention, the expression of the second regulatory factor, the Cre recombinase, is inhibited in the lox-cre-dependent method after the addition of an inhibitor, which enables expression of the reporter genes in the first place. The use of a recombinase in the present method enables inhibitor detection according to an "all or nothing" principle, whereby, according to the present invention, the positive detection of reporter genes after the addition of inhibitor proves to be particularly suitable and enables very sensitive detection.

Example 5

This example represents the configuration shown in FIG.

Gene arrangements are provided which code for two interacting proteins, one arrangement coding for a protein which contains a DNA-binding domain, a domain which binds to a further second protein and an activating domain, and a further arrangement for one another second protein which contains at least one domain interacting with the first protein (FIG. 5a).

Furthermore, a repressor gene arrangement is provided with one or more binding sites on the DNA (target binding site) for the transcription regulator. 3. A reporter gene arrangement as described under Example 1, point 3 is also provided.

a. The transcription regulator is composed of the two proteins described above, an active transcription regulator being present only when the protein-protein interaction of the two proteins is undisturbed (see FIG. 5a). If there is no inhibitor, an undisturbed one is found

Protein-protein interaction (Target I - Target II) takes place and an active transcription regulator is available. The active transcription regulator binds to its corresponding binding site on the DNA (Target I binding site), which is responsible for the

Repressor gene encodes, and causes expression of the repressive gene. The repressor binds to the repressor binding site in the promoter region of the reporter genes and represses expression of the reporter genes. Since Leu2 and / or LacZ are not expressed, there is no growth on leucine-deficient medium, and there is no blue color after growth on medium containing X-Gal.

b. If an inhibitor is added to the experimental set-up, e.g. the inhibitors mentioned above, for example peptides, nucleic acids, carbohydrates or other chemical substances, the protein-protein

Interaction of the proteins of the transcription regulator inhibited. There is now no active transcription regulator, because if the interaction of the proteins is inhibited, the transcription regulator is not in the active form

(Conformation) is present. The DNA binding domain of a protein of the

Transcription regulator can, depending

Test condition and added inhibitor, bind to the non-binding DNA section or not. Due to the inhibited protein-protein interaction, there is no active transcriptional regulator in any case. Due to the inactive transcription regulator, there is no expression of the repressive morning.

Since there is no repressor, after binding another one

Transcription factor TF, which expresses reporter genes Leu2 and / or LacZ. There is now growth of the host cells on leucine-deficient medium or a change in color (blue staining) after growth on medium containing X-Gal is detectable. According to the present invention, the addition of an inhibitor via inhibition of the second regulatory factor (repressor) leads to expression of the reporter genes.

Example 6

This example relates to the configuration shown in FIG. 6.

1. There are gene arrangements, as described in Example 5, provided for interacting

Coding proteins that form an active transcription regulator via a protein-protein interaction.

2. Furthermore, a gene arrangement is provided which is suitable for the sequence-specific recombinase Cre des Coliphagen Pl coded. This recombinase corresponds to the second regulatory factor.

a. As also described under Example 5, if there is no inhibitor, there is an undisturbed protein-protein interaction between the

Proteins forming transcription regulator take place, whereby an active transcription regulator is present. The active transcription regulator binds to a binding site on the DNA, this DNA being the one for the Cre-

Contains Cre gene encoding recombinase. After binding the transcription regulator, the Cre recombinase is expressed. This recombinase eliminates or inverts the reporter genes via recombination processes, interacting with the lox P sequences which flank the reporter genes. No functional reporter genes are expressed either after elimination or after inversion. Accordingly, without adding one

Inhibitors do not grow on leucine-deficient medium, nor do they turn blue when growing on medium containing X-Gal.

b. After adding an inhibitor to the test batch, the protein-protein interaction of the Proteins, eg via confirmation change, inhibited the transcription regulator. There is therefore no active transcription regulator. The DNA-binding domain of a protein of the transcription regulator binds to the relevant DNA section or not, depending on the test conditions and the inhibitor added. Due to the inhibited protein-protein interaction, there is no active transcriptional regulator in any case. Due to the lack of an active transcription regulator, no transcription regulator binds to the binding site on the DNA which codes for the Cre recombinase. The Cre recombinase is therefore not expressed. Accordingly, the reporter genes Leu2 and / or LacZ are neither eliminated nor inverted. A lack of recombinase thus leads to expression of the reporter genes, which enables growth of the cells in leucine-deficient medium or a blue coloration in medium containing X-Gal.

According to the present invention, the expression of the second regulatory factor, the Cre recombinase, is inhibited in the lox-Cre-dependent method after the addition of an inhibitor, which enables expression of the reporter genes in the first place. The resulting advantages have already been described in Example 4b.

Example 7

This example represents the configuration shown in FIG. 7. 1. Gen arrangements are provided which code for two interacting proteins Target II and Target III and contain a UAS sequence and a TATA box, an arrangement coding for a protein Target III which comprises a protein from an anchoring section and a first one Is a protein component, and a further arrangement encodes a protein target II, which is a fusion protein of an inhibiting section and a second protein component (FIG. 7a).

2. A gene arrangement for a transcription factor Target I is provided which can be inhibited.

3. Furthermore, a repressor gene arrangement is provided with one or more binding sites on the DNA (target binding site) for the transcription factor.

4. A reporter gene arrangement as described under Example 1, point 3 is also provided.

a. In the case of an undisturbed protein-protein interaction of the two proteins, the inhibiting section of the first protein cannot interact with the transcription factor, so there is an active transcription factor (see FIG. 7a). If there is no inhibitor, an undisturbed protein-protein interaction takes place and an active transcription factor is present. This active transcription factor binds to its corresponding binding site on the DNA, which codes for the repressor gene, and causes expression of the repressor gene. The

Repressor binds to the repressor binding point in the Promoter region of the reporter genes and represses expression of the reporter genes. Since Leu2 and / or LacZ are not expressed, growth does not take place on leucine-deficient medium, and there is no blue color after growth on medium containing X-Gal.

If an inhibitor is added to the experimental set-up, e.g. the inhibitors mentioned above, for example peptides,

Nucleic acids, carbohydrates or other chemical substances, the protein-protein interaction of the proteins Target II and Target III is inhibited. The protein with the inhibiting section Target II is released and binds to the transcription factor Target I, whereby this is inhibited. There is now no active transcription factor.

Because of the inhibited protein protein

There is no interaction with an active transcription regulator. Due to the inactive transcription factor, there is no expression of the repression morning.

Since there is no repressor, the reporter genes Leu2 and / or LacZ are expressed after binding a corresponding further transcription factor TF. There is now growth of the host cells on leucine-deficient medium

Expression of the reporter genes. Example 8

This example relates to the configuration shown in FIG. 8.

1. Gen arrangements as described under Example 7 are provided which code for interacting proteins.

2. A gene arrangement for a transcription factor is provided (Target I).

3. Furthermore, a gene arrangement is provided which is suitable for the sequence-specific recombinase Cre des

Coliphagen Pl coded. This recombinase corresponds to the second regulatory factor.

4. A reporter gene arrangement with the reporter genes Leu2 and / or LacZ is also provided, the structure of both reporter genes in the present method having the same regulatable promoter. This promoter contains UAS elements, to which an endogenous activator preferably binds, and a TATA box. The reporter genes have flanking lox P sequences as recombination elements.

a. As also described under Example 7, if there is no inhibitor, there is an undisturbed protein-protein interaction between the

Proteins instead, which is an active transcription factor. The active transcription factor Target I binds to a binding site on the DNA, this DNA containing the Cre gene coding for the Cre recombinase. After binding the transcription factor the Cre recombinase is expressed. This recombinase eliminates or inverts the reporter genes via recombination processes, interacting with the lox P sequences which flank the reporter genes. No functional reporter genes are expressed either after elimination or after inversion. As a result, there is no growth on leucine-deficient medium without the addition of an inhibitor, and no blue coloration either

Growth on medium containing X-Gal.

After adding an inhibitor to the test batch, the protein-protein interaction of the proteins Target II and Target III is inhibited. The

Protein with the inhibiting section Target II is released and interacts with the transcription factor, whereby this is inhibited. There is therefore no active transcription factor. Depending on the experimental conditions, the DNA-binding domain of the transcription factor binds to the relevant DNA section or not. In no case is there an active transcription factor due to the inhibited protein-protein interaction. As a result of the lack of an active transcription factor, no transcription factor binds to the binding site on the DNA which codes for the Cre recombinase. The Cre recombinase is therefore not expressed. The reporter genes Leu2 and / or

LacZ is neither eliminated nor inverted. A lack of recombinase thus leads to expression of the reporter genes, which enables growth of the cells in leucine-deficient medium or blue staining in X-Gal-containing medium

Medium occurs. According to the present invention, the expression of the second regulatory factor, the Cre recombinase, is inhibited in the lox-cre-dependent method after the addition of an inhibitor, which enables expression of the reporter genes in the first place.

Example 9

The operation of the process is explained in more detail using the example of the Tet repressor and its specific inhibition by tetracycline.

This example represents the configuration shown in FIG. 9.

1. A gene arrangement is provided which codes for the first regulatory factor ADl-Tet. This regulatory factor binds to DNA areas

DNA (Tet binding site) that codes for the second regulatory factor, the repressor LexA-GST.

2. Furthermore, a repressor gene arrangement of the repressor LexA-GST is provided with a binding site on the DNA (Tet binding site) for the first regulatory factor ADl-Tet mentioned under 1.

3. A reporter gene arrangement with the reporter genes Leu2 and / or LacZ is also provided, the structure of both reporter genes in the present method having the same regulable promoter. This promoter consists of so-called UAS elements (upstream activation sequence), to which an endogenous activator (further Transcription factor Gal4) binds from one or more LexA binding sites to which a LexA-GST can bind and a TATA box for the initiation of the basal transcription.

a. The expression of the fusion clone ADl-Tet (first regulatory factor) stimulates the expression of the repressor LexA-GST (second regulatory factor). This repressor protein in turn is repressed by the

Binding to the LexA binding site of the LacZ or LEU2 promoter described in FIG. 9 is the Gal4-activated expression of the reporter genes LacZ and Leu2 which is independent of the first regulatory factor. Since the Gal4-activated expression of

Reporter genes inhibited by glucose and only stimulated to galactose, these yeasts cannot grow on leucine-deficient media and remain white on X-gal-containing media (Table 1). If the medium has galactose as a sugar source, the Gall-LexA promoter is further inhibited by the ADl-Tet-dependent expression of the LexA-GST repressor: the yeast colonies remain white and do not grow on leucine-deficient media.

b. Depending on the increasing tetracycline concentration (Figure 9 and Tables 1 and 2), the binding of the ADl-Tet protein to the Tet-dependent promoter of the LexA-GST repressor is specifically inhibited. The increasing tetracyklin concentration used here does not inhibit the general growth of the yeast. The second regulatory factor is no longer able to inhibit the activity of the reporter system. The yeast cells are now using galactose as a sugar source and X- Gal as a substrate for the blue reporter system and can grow on leucine-deficient media. In contrast, glucose-containing medium inhibits the activity of the endogenous transcription factor Gal4. The yeasts do not grow on leucine-deficient media and remain white on media plates containing X-Gal. Other antibiotics such as ampicillin, chloramphenicol, kanamycin and carbenicillin have no such repressing effect on the first regulatory factor ADl-Tet (Table 1). On plates containing glucose, Gal4 is inhibited regardless of the first and second regulatory factors, which is why the yeasts on such media do not grow and remain white.

Table 1:

Add tetracycline growth to LEU blue stain on X-Gal

Glucose galactose glucose galactose white white

+ + -H- white blue

Table 2: (only the results of the plates containing galactose are shown)

a) Concentrations used [μg / ml]

Antibiotic used 0 5 50 250

Tetracyklin - - / white - - / white - - / white + + / blue

Ampicillin - - / white - - / white - - / white - - / white

Kanamycin - - / white - - / white - - / white - - / white

Carbenicillin - - / white - - / white - - / white - - / white

Chloramphenicol - - / white - - / white - - / white - - / white

Growth on LEU / blue coloring on X-Gal

b) Concentrations used [μg / ml]

Antibiotic used 0 5 50 100 150 200 250 300

- - / - - / - - / - - / - - / + + / + + / + + /

Tetracyklin white white white white white blue blue blue

Growth on LEU / blue coloring on X-Gal

c) Evidence of the functioning of the individual elements in the assay

ADl-Tet as a transcription factor:

The following assay was carried out to demonstrate that the fusion clone ADl-Tet (first regulatory factor) expresses functionally in the yeast, transports it to the nucleus and efficiently binds it to the DNA consensus sequence and from there can activate the transcription of a gene : ADl-Tet is expressed on media plates containing galactose and is therefore able to bind to the Tet binding site of a corresponding promoter in front of the LacZ gene and from there to activate the expression of the reporter gene LacZ. This in turn can be demonstrated by converting the colorless substrate X-Gal contained in the medium into a blue indigo dye. Since glucose inhibits the expression of the ADl-Tet protein, there is no expression of the reporter gene LacZ. The colonies remain white.

Tetracycline as a specific inhibitor of the transcription factor Tet-AD

In order to demonstrate that tetracycline can specifically inhibit the binding activity of the ADl-Tet protein, increasing concentrations of the antibiotic were added to the media plates:

Table 3:

Concentrations used tetracycline [μg / ml]

0 5 50 100 150 200 250 300

Blue coloring on X-Gal blue blue blue blue blue white white white

Galactose-containing + + -H- -H- + + + + + + + + + + plates with leucine in the medium

Glucose-containing + + ++ -H- + + + + + + + + + + plates with leucine in the medium

As the experimental data show, depending on the increasing tetracycline concentration (Table 3) the binding of the ADl-Tet protein to the Promoter of the reporter gene LacZ can be specifically inhibited. At a concentration of 200 μg / ml tetracycline in the medium, the yeast cells remain white. The general growth of the yeast cells is not significantly affected by this, as the results on the plates containing glucose and galactose show (the growth is represented by "++").

LexA-GST as a repressor

The following assay was carried out to demonstrate that the second regulatory factor (LexA-GST) expresses functionally in the yeast, is transported into the nucleus and is efficiently bound there to the LexA consensus sequence and from there can inhibit the transcription of a gene.

In contrast to the negative control CTF2 (a member of the NFI family), the yeast clones remain white on media containing X-Gal and galactose. This means that the LexA-GST fusion clone binds between the Gal4 binding site and the start of transcription of the reporter gene and thus inhibits the activity of the endogenous yeast transcription factor Gal4. CTF2, on the other hand, is not able to

Recognize binding sites and can not repress the activity of Gal4. The reporter gene LacZ is expressed and the colonies turn blue (Altmann et al., Proc. Natl. Acad. Sei. (1994) 91, 3901-3905).

Example 10

Identification of specific peptide inhibitors of CTF7

To inhibit peptide sequences against CTF7, a member of the NFI- Family (Altmann et al., Proc. Natl. Acad. Sci. (1994) 91, 3901-3905), the following assay (Fig. 10) was carried out.

This example represents the configuration shown in FIG. 10.

1. A gene arrangement is provided which codes for a first regulatory factor LexA-CTF7. The corresponding plasmid pEG-CTF7 contains the gene arrangement for the first regulatory factor LexA-CTF7. This first regulatory factor LexA-CTF7 binds to DNA regions of a DNA (LexA binding site) which codes for a second regulatory factor Tet-GST.

2. Furthermore, a gene arrangement for the second regulatory factor Tet-GST is provided with a binding site on the DNA (LexA binding site) for the first regulatory factor LexA-CTF7 mentioned under 1.

3. A reporter gene arrangement with the reporter genes Leu2 and / or LacZ is also provided, both repσrtergenes in the present process from

Structure have the same adjustable promoter. This promoter is made up of so-called UAS elements (upstream activation sequence), to which an endogenous activator (another transcription factor Gal4) preferably binds, from a tet

Binding site to which Tet-GST can bind and a TATA box for the initiation of basal transcription.

a. If no inhibitor is added to the experimental set-up, there is an active first regulatory factor LexA-CTF7, which positively regulates the expression of the Tet-GST gene. Tet-GST binds to the above-mentioned Tet-GST binding site and represses the expression of the reporter gene LacZ and / or Leu2, ie there is no growth of the host organisms, no color reaction can be detected.

After adding a Trx peptide, the activity of the first regulatory factor LexA-CTF7 is inhibited. This is due to an inhibition of the interaction of LexA-CTF7 with the corresponding binding site on the DNA or an inhibition of the activation domain. There is no expression of the Tet-GST gene. There is therefore no Tet-GST available, and the reporter gene LacZ and / or Leu2, which is connected downstream of the promoter, is strongly expressed by an inducible endogenous transcription factor Gal4. As a result, the yeasts grow in leucine-deficient media, and after growth in medium containing X-Gal, a color change (blue) occurs.

According to the present invention, the addition of the Trx peptide leads to an expression of the

Reporter gene / the reporter genes, in that the second regulatory factor Tet-GST is not repressed or reduced.

The test is carried out in detail as follows:

The yeast strain INVScl was transformed with the plasmids shown in FIG. 10 and the

Transformation approach on a minimal medium plate containing glucose (20 x 20 cm; uracil, Streaked with tryptophan and histidine for selection on the plasmids).

The vector pEG-CTF7 (corresponds to the first regulatory factor) and was produced according to Altmann et al. (Proc. Natl. Acad. Sei. (1994) 91, 3901-3905).

pYES-Leu2 / 34-TetGST (plasmid which encodes the second regulatory factor TetGST and which

Reporter system included).

The plasmid pYES2 served as the starting vector. The LexA binding sites oligos were ligated into the Xhol site of the inactive Gall / GallO promoter, the entire promoter was isolated with BamHI and cloned into the polylinker of the plasmid pYES2 together with the fusion clone Tet-GST. The Gall promoter had previously been deleted via the Spei interfaces. In the Nhel interface of this construct treated with Klenow polymerase, the LacZ fragment isolated via BamHI, also treated with Klenow, or the Leu2 fragment obtained via PCR was cloned. The was reconstituted in the BamHI interface

Ligation of the Gal / Tet promoter. This was obtained by cloning Tet oligos and then ligating Gal4 binding sites into the Xhol site of the inactive Gall / 10 promoter.

pYES2 (TRPl) TRX oligo pool (construct expressing the peptide pool; complexity approx. 10 ⁵ )

By ligating the Trpl gene into the pYES2 vector linearized with Apal and Nhel, the Ura3 gene destroyed and the plasmid selectable for tryptopic deficiency. The Trx gene isolated via PCR was then cloned into the EcoRI, Xhol linearized vector. The pool fragments were ligated via the Rsrll

Interface of the Trx construct.

The approximately 30,000 colonies were combined, shaken for 5 hours in YPG medium (complete medium containing galactose for yeasts) and spread on selection plates. The selection plates had galactose as a carbon source and were deficient in leucine, uracil, tryptophan and histidine. 8 colonies (A, B, C, D, E, F, G, H) had grown over a period of 2 to 5 days (Table 4). For further specification, the plasmids coding for the inhibiting peptides were isolated from these and expressed together with CTF7 and Tet-GST in yeast. This time the LacZ gene was on the reporter plasmid instead of the Leu2, so that the inhibitory effect of the peptides could be investigated by the formation of a second phenotype (Table 4). Furthermore, the plasmids expressing the peptides were transformed with the two reporter systems (LacZ and Leu2) and a transcription factor LexA-TAi different from CTF7 in yeasts. In this way, peptides that specifically inhibit CTF7 can be distinguished from non-specific inhibitions.

Table 4:

Blue staining on plates containing X-Gal

Identified growth on inhibition of leucine-LexA-TAl inhibitors on deficient glucose galactose galactose plates

A + - + -

B + + + n.b.

C + + + n.b.

D + - + +

E + - + -

F + - + -

G + - + -

H + - + +

The inhibitors B and C also turned blue on plates containing glucose, ie independently of the galactose-induced TRX peptide expression. This means that no inhibiting peptides which cause the blue coloration are expressed in the corresponding clones. Of the peptides A, D, E, F, G and H, D and H turned out to be unspecific because they were also able to inhibit the LexA-TA- ^ domain. The inhibitors A, E, F and G, however, were only able to repress the activity of CTF7 (n.b .: not determined).

Example 11

Identification of specific inhibitors of the protein-protein interaction LexA-CTF2 / AD1-TIM The assay described in Figure 11 is performed to screen inhibitory peptide sequences against the yeast-active protein-protein interaction LexA-CTF2 and TIM-AD (Altmann (1994) dissertation). LexA-CTF2 is a fusion construct from the bacterial repressor LexA and CTF2, a member of the NFI family. For this purpose, the yeast strain INVScl is transformed with the plasmids shown in FIG. 11 and the transformation mixture is spread on a minimal medium plate containing glucose (20 × 20 cm; uracil, tryptophan and histidine deficient for selection on the plasmids).

This example represents the configuration shown in FIG. 11.

1. Gene arrangements are provided which code for two interacting hybrid proteins of the transcription regulator and contain a UAS sequence and a TATA box, an arrangement for a LexA-CTF2, a fusion construct from the bacterial repressor LexA and CTF2, a member of the NFI Family (Altmann et al., Proc. Natl. Acad. Sei. (1994) 91, 3901-3905) and a further arrangement for a hybrid protein TIM-AD (Altmann (1994), dissertation).

pSH-CTF2 / TIM (corresponds to the first regulatory factor) is the corresponding plasmid which encodes the first regulatory factor LexA-CTF2 / ADl-TIM.

2. pYES-Leu2 / 34-TetGST (plasmid which encodes the second regulatory factor TetGST and contains the reporter system). 3. pYES2 (TRPI) TRX-01igo pool (construct expressing the peptide pool; complexity approx. 10 * 5).

a. The first regulatory factor LexA-CTF2 / ADl-TIM consists of two, as described above

Fusion proteins LexA-CTF2 and AD1-TIM together, an active first regulatory factor pSH-CTF2 / TIM being present only when the protein-protein interaction of the two fusion proteins is undisturbed (see FIG. 11a). If there is no inhibitor, there is an undisturbed protein-protein interaction and an active transcription regulator is present. The active transcription regulator LexA-CTF2 / ADl-TIM binds to its corresponding binding site on the DNA

(LexA binding site), which codes for the Tet-GST gene, and causes expression of Tet-GST. Tet-GST binds to the corresponding binding site (Tet-GST binding site) in the promoter region of the reporter genes and represses expression of the

Reprotergenes. Since Leu2 and / or LacZ are not expressed, there is neither growth on leucine-deficient medium nor a blue color after growth on medium containing X-Gal.

b. If an inhibitor is added to the experimental set-up, e.g. a Trx peptide, the protein-protein interaction of the fusion proteins LexA-CTF2 and AD1-TIM is inhibited. There is now no active first regulatory factor.

Because of the inactive first regulatory

Factor LexA-CTF2 / ADl-TIM there is no expression of the Tet-GST gene (repressor gene). Since there is no repressor Tet-GST, after binding of a further transcription factor Gal4, which expresses reporter genes Leu2 and / or LacZ.

The test is carried out in detail as follows:

The approximately 35,000 colonies were collected, shaken for 5 hours in YPG medium (full galactose-containing medium for yeasts) and spread on selection plates. The selection plates had

Galactose as a carbon source and were deficient in leucine, uracil, tryptophan and histidine. 6 colonies (A, B, C, D, E, F) have grown over a period of 2 to 5 days. For further specification, the plasmids coding for the inhibiting peptides were isolated from these and expressed together with LexA-CTF2, TIM-AD and Tet-GST in yeast. This time, instead of the Leu2, the LacZ gene was on the report plasmid, so that the inhibitory effect of the peptides could be investigated by the formation of a second phenotype (Table 5). Furthermore, the plasmids expressing the peptides were transformed with the two reporter systems (LacZ and Leu2) and the transcription factor LexA-TAl in yeast. In this way the TIM-CTF2 interaction specifically inhibiting peptides could be separated from non-specific ones.

Table 5:

Blue staining on plates containing X-Gal

A + + + n.b.

B + - + -

C + + + n.b.

D + - + +

E + - + +

F + - + -

The inhibitors A and C also turned blue on plates containing glucose, ie independently of the galactose-induced TRX peptide expression. This means that no inhibiting peptides which cause the blue coloration are expressed in the corresponding clones. Of the peptides B, D, E and F, D and E turned out to be unspecific, since they were also able to inhibit the LexATTAi domain. In contrast, inhibitors B and F were not able to repress ADl-Tet, so they are inhibitors for the CTF2-TIM protein-protein interaction.

In experiments 1 to 11 shown, there are qualitative and / or quantitative differences in inhibitory activity for different inhibitors, i.e. in addition to a complete inhibition of the first regulatory factor, gradual weakening of the activity of the first regulatory factor is also possible. Accordingly, in addition to full expression, gradual enhancements are also possible and detectable in the expression of the reporter genes.

Claims

claims

1. Method for determining the activity of at least a first regulatory factor, which via the

Activity of at least one reporter system is detectable, characterized by the following

Steps:

a. Providing at least one reporter system with at least one first gene arrangement which has at least one reporter gene,

b. Providing at least one second gene arrangement which codes for at least one second regulatory factor, and interaction of the second regulatory factor with components of the reporter system, thereby acting on the activity of the reporter system,

c. Influence preferably on the activity of the second regulatory factor by the at least one first regulatory factor, and

d. Detection of the activation of the at least one reporter system by adding at least one inhibitory component via the interaction of the first and second regulatory factors.

2. The method according to claim 1, characterized in that the first regulatory factor interacts with DNA sections of the coding for the second regulatory factor second gene arrangement, thereby acting on the expression of the second regulatory factor.

3. The method according to any one of claims 1 or 2, characterized in that the second regulatory factor binds to DNA sections of the reporter system.

4. The method according to any one of the preceding claims, characterized in that the at least one first regulatory factor contains one or more regulatory components.

5. The method according to claim 4, characterized in that the one or more regulatory components are one or more regulatory proteins.

6. The method according to claim 5, characterized in that the regulating protein or the regulating proteins are one or more transcription regulators.

7. The method according to claim 6, characterized in that the transcription regulator or the transcription regulators are one or more transcription factors.

8. The method according to any one of claims 5 to 7, characterized in that the regulating protein or the regulating proteins modify the at least one second regulatory factor.

9. The method according to any one of the preceding claims, characterized in that the modification of the at least one second regulatory factor is carried out by kinasing, dephosphorylation, cleavage, refolding or change of conformation.

10. The method according to any one of the preceding claims, characterized in that the addition of the inhibitory component acts on the interaction between at least two components which together form the first regulatory factor, and / or on the activity of the at least one first regulatory factor.

11. The method according to claim 10, characterized in that the interaction between at least a first regulatory factor and a DNA section of the at least one second gene arrangement is acted on by adding the inhibitory component.

12. The method according to any one of the preceding claims, characterized in that the influencing of said activity of the first regulatory factor by inhibitory components has an effect on the gene expression of the second

Gene arrangement leads.

13. The method according to any one of claims 5 to 12, characterized in that an inhibitor via an inhibition of the activity of the regulating protein to a

Inhibition of expression of the second gene assembly leads.

14. The method according to any one of claims 1 to 9, characterized in that the interaction between at least two components is acted upon by the addition of the inhibitory component, the at least one first component being or containing a regulatory component.

15. The method according to claim 14, characterized in that the regulatory component is or contains a protein component.

16. The method according to claim 15, characterized in that the at least one first protein component is a fusion protein.

17. The method according to any one of claims 14 to 16, characterized in that the regulatory component is or contains an inhibitory component.

18. The method according to any one of claims 14 to 17, characterized in that the at least one second component is or contains at least one protein component.

19. The method according to claim 18, characterized in that the at least one second protein component is at least one fusion protein.

20. The method according to any one of claims 14 to 19, characterized in that the at least one second component has an anchoring function.

21. The method according to claim 20, characterized in that via an interaction of the at least two protein components, these protein components are localized in the cytoplasm.

22. The method according to claim 20, characterized in that via an interaction of the at least two protein components, these protein components are located on or in a membrane.

23. The method according to any one of claims 14 to 22, characterized in that by adding an inhibitory component via an inhibition of the interaction between the at least two components, the at least one first component, which contains the inhibitory section, is released and with the transcription activating Gene arrangement factor interacts for the second regulatory factor.

24. The method according to claim 23, characterized in that the transcription-activating factor is inhibited by the released inhibitory factor, whereby the expression of the second gene arrangement is inhibited.

25. The method according to any one of the preceding claims, characterized in that microorganisms, preferably bacteria, or eukaryotic cells, preferably yeasts, are used as host organisms.

26. The method according to claim 25, characterized in that the bacterial strain Escherichia coli or the yeast strain Saccharomyces cerevisiae is used.

27. The method according to any one of the preceding claims, characterized in that at least one gene product of the at least one reporter gene is detectable.

28. The method according to claim 27, characterized in that the detection of the gene product is carried out by one or more changes in the phenotype of the host organism.

29. The method according to claim 28, characterized in that the at least one gene product of the reporter gene enables cell growth of the host organisms in deficient medium.

30. The method according to claim 29, characterized in that the gene product of the reporter gene is Leu2 and enables cell growth in leucine-deficient medium.

31. The method according to claim 28, characterized in that the gene product of the reporter gene LacZ can convert substrates in a measurable color reaction.

32. The method according to any one of the preceding claims, characterized in that the said

Gene arrangements are arranged on different vectors.

33. The method according to any one of claims 1 to 31, characterized in that said gene arrangements are arranged on the same vector.

34. The method according to any one of claims 32 or 33, characterized in that the vectors are plasmids.

35. The method according to any one of claims 32 to 34, characterized in that one or more vectors with one or more gene arrangements are integrated into the host genome.

36. The method according to any one of the preceding claims, characterized in that the second gene arrangement codes for at least one repressor.

37. The method according to claim 36, characterized in that the repressor acts on the expression of the at least one reporter gene.

38. The method according to any one of claims 36 or 37, characterized in that the repressor acts by binding to components of the reporter system.

39. The method according to claim 38, characterized in that the binding of the repressor to components of the

ReporterSystems can be regulated by additional agents.

40. The method according to any one of claims 36 to 39, characterized in that a LacZ in a reporter plasmid

Leu2 gene arrangement and the repressor gene arrangement are integrated.

41. The method according to any one of claims 1 to 35, characterized in that the second gene arrangement codes for at least one recombinase and the reporter systems contain recombination elements.

42. The method according to claim 41, characterized in that the recombinase eliminates or inverts at least one reporter gene via recombination processes.

43. The method according to any one of claims 41 or 42, characterized in that the recombinase is the sequence-specific recombinase Cre of Coliphagen P1 and which has at least one reporter gene flanking lox P sequences as recombination elements.

44. The method according to any one of claims 6 to 13 and 25 to 43, characterized in that the Transcription regulator contains at least two hybrid proteins, the hybrid proteins being encoded by a third gene arrangement.

45. The method according to claim 44, characterized in that a first hybrid protein is a fusion protein from a DNA binding domain and a first protein component and a second hybrid protein is a fusion product from an activation domain of the transcription regulator and a second

Protein component is, the active transcription regulator being formed by binding between the two protein components.

46. The method according to claim 45, characterized in that there is an effect on the interaction of the hybrid proteins via inhibitory components.

47. The method according to any one of claims 15 to 24, characterized in that the at least two protein components are encoded by a fifth gene arrangement.

48. The method according to any one of claims 14 to 24, characterized in that the at least one first protein component contains a section which interacts with the at least one second protein component, a section which interacts with a transcription factor and an inhibitory section, only after inhibition the interaction of the bond between the at least two protein components, the inhibitory first protein component inhibits the expression of the at least one second regulatory factor.

49. The method according to any one of claims 14 to 16, characterized in that the at least one first regulatory component is a transcription regulator or transcription factor of the gene arrangement of the second regulatory factor, which reduces or inactivates its activity after inhibiting the interaction with the at least one second protein component which inhibits the expression of the second regulatory factor.

50. The method according to any one of the preceding claims, characterized in that these inhibitory components are natural substances such as peptides, nucleic acids and carbohydrates or other chemical substances.

51. The method according to claim 50, characterized in that the inhibitory components are mutagenesis-modified components of the at least one first regulatory factor.

52. The method according to any one of the preceding claims, characterized in that at least one further fourth gene arrangement is provided for the expression of peptides.

53. The method according to any one of claims 44 to 46 and 50 to 52, characterized in that by the

Action on the interaction of the at least two hybrid proteins of the transcription regulator, the expression of at least one repressor gene is controlled, as a result of which a change in the expression of the at least one reporter gene takes place.

54. The method according to claim 53, characterized in that by inhibiting the interaction of the hybrid proteins of the transcription regulator, the expression of the repressor gene is inhibited and the at least one reporter gene is expressed.

55. The method according to any one of claims 44 to 46 and 50 to 52, characterized in that the effect on the interaction of the at least two hybrid proteins of the transcription regulator

Expression of at least one recombinase is controlled, whereby a change in the expression of at least one reporter gene takes place.

56. The method according to claim 55, characterized in that by inhibiting the interaction of the hybrid proteins of the transcription regulator, the expression of the recombinase is inhibited and the at least one reporter gene is expressed.

57. The method according to any one of claims 14 to 24 and 47 to 49, characterized in that the expression of at least one repressor gene is controlled by the action on the interaction of the at least two protein components, whereby one

The expression of the at least one reporter gene is changed.

58. The method according to claim 57, characterized in that by inhibiting the interaction of the

Protein components, the expression of the repressor gene is inhibited and the at least one reporter gene is expressed.

59. The method according to any one of claims 14 to 24 and 47 to 49, characterized in that by the Influence on the interaction of the at least two protein components, the expression of at least one recombinase is controlled, as a result of which a change in the expression of at least one reporter gene takes place.

60. The method according to claim 59, characterized in that by inhibiting the interaction of the protein components, the expression of the recombinase is inhibited and the at least one reporter gene is expressed.

61. The method according to any one of claims 41 to 60, characterized in that gene arrangements for at least one repressor and at least one recombinase are simultaneously provided in a host organism.

62. The method according to claim 61, characterized in that, depending on the test conditions set, the repressor or the recombinase or both are expressed.

63. Use of one of the methods according to one of claims 1 to 62 for the determination of inhibitory substances which can preferably be used as lead structures.

64. Use according to claim 63, characterized in that the inhibiting substances are peptides.

65. Use according to one of claims 63 or 64, characterized in that the inhibiting substances and peptides are used for the development of therapeutic agents.