DE19502584C2 - Method for determining the activity of a regulatory factor and use of this method - Google Patents

Description

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

In living cells, metabolic performance is either continuously or only in certain development phases or based on external signals. When through Hormones mediated signal transmission, for example corresponding signals about interactions between Proteins from the outer cell membrane into the cell interior transported in to there, for example in the cell nucleus, in a request for division to be implemented. Become disrupting these interactions, this can make a healthy cell unbalance and growth disorders to lead. So z. B. a degenerate cell to one Cancer Grow Up and About the Formation of Metastases the deadly tumor all over the body to distribute. Viruses such as HIV (Human Immunodeficiency Virus) or HPV (Human Papilloma Virus) intervene in the natural Processes of the cell and abuse it to itself reproduce in order to then infect further cells.

It is obvious that such interactions be deliberately intervened in the events of a cell can, provided the corresponding proteins are known and a simple test are accessible. In the past years such tests have been developed. They are ultimately based insist that a simple biochemical reaction by a color reaction detectable and thousands of times is feasible at the same time from such Interaction between proteins is made dependent. So-called in vivo assays allow these interactions e.g. B. in yeast cells that are easy to grow and also for  the formation of human proteins are suitable (Brent, R. et al., PCT publication WO 92/05286; Fields, S. et al., U.S. 5,283,173).

The transcription of protein coding genes is carried out by one Multiprotein complex consisting of Pol II and a series specific and general transcription factors initiated. A variety of these factors have occurred in recent years have been isolated and characterized, making one 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. Chem. (1992) 267, pp. 2786-2793).

In addition to the basal transcription machinery, play above all the DNA-binding transcription factors are crucial Role in stimulated gene expression.

The three characteristic features of Transcription activators, such as proto-oncogenes or viral ones Transcription factors and protein-protein interactions in signal chains or multiprotein complexes, which they too make particularly suitable targets for examinations their high diversity, their specificity and their possible Role in the development of diseases. So z. B. more as 300 gene-specific transcription factors to date and it is believed that approximately 3000 more such factors are encoded by the human genome. Similar to how receptors on the cell surface resemble practically no factor and no protein protein Interaction of exactly the other. Every protein offers one unique surface and thereby represents a unique Target.  

The DNA binding and the stimulating activity must but not localized on a polypeptide chain (Weston et al., Cell (1989) 58, pp. 85-93). The division of this Both properties allow, for example, the investigation an interaction between two proteins X and Y, where the DNA-binding part on a protein X and the transcription-active part localized on a protein Y. (Fields et al., Nature (1989) 340, pp. 245-246). The specific inhibition of this interaction results in Loss of the stimulating activity of this element.

So far, methods are known to detect inhibitors which inhibit the biological activity of oncoproteins (WO 92/05286). These procedures are used to identify inhibitory components and classify by the ability of such a component the expression of reporter genes is examined influence. According to the aforementioned PCT publication become fusion genes to perform this procedure provided which code for fusion proteins. This Fusion proteins bind to a binding site on the DNA, whereby encodes this DNA for the reporter genes, thus mediating the Expression of the reporter gene. Will the expression of the Reporter gene examined, there is a decrease in reporter gene Expression indicative of a component that the Inhibits activity of the fusion proteins.

Inhibitors are detected using the mentioned procedures only through an acceptance of Expression of the reporter genes in question, d. H. it deals a negative proof. The well-known detection system is disadvantageous in that it may be the Reduced sensitivity of a test system. Is z. B. a Reporter gene is in principle only weakly expressed, so are Inhibitor addition and thus further decreasing expression often no clear statements can be made. These disadvantages  are due to the fact that an added inhibitory Component inhibits a transcription factor that the Expression of reporter genes affected, i. H. It is about for a direct functional connection of inhibitor and Reporter gene. In particular, a missing expression is after Inhibitor addition is disadvantageous because of the experimental procedures are often based on selection of organisms, as follows briefly explained. So z. B. the growth of cells after If inhibitor addition is investigated, they are growing inhibited cells and do not need to be examined Experiments are proven. A screening of many different potential inhibitors is therefore not possible. The lack of expression of reporter genes after The addition of inhibitors can also influence the influence other factors, such as the Inhibitors on factors of translation and Replication mechanisms and the cell cycle. Thus the Often, the place of action of the inhibitor cannot be clearly defined, resulting in a lack of specificity of the previous Detection systems result.

The object of the present invention is therefore a Provide procedures that the aforementioned Does not have disadvantages, the fast, clear and enables specific statements about test results as well improves the sensitivity of test systems.

The invention solves this problem by the independent Claim 1 specified method and the use according to Claim 63. Further preferred configurations, Aspects and details of the method according to the invention are in dependent claims 2 to 62 and 64, the Drawings, tables and the preferred embodiments spelled out. The present invention represents an essential improved detection system for the activity of a regulatory factor. With the specified  A procedure can also be carried out in the living organism inhibitory component by expression or enhanced Expression of a reporter system can be demonstrated without that the disadvantages of known methods occur.

This significantly increases the sensitivity of the test system and enables the screening of inhibitor libraries of great complexity (over 10 ⁹ different molecules).

This is a new principle for screening for inhibitors and chemicals that modify activities, used. The assay can also be used for identification so far unknown interactions are used.

This is essential for the method according to the invention Provide at least a second regulatory Factor. The procedure described below will preferably carried out in host organisms, however Appropriate procedures should not be excluded also perform outside of an organism. As Host organisms become microorganisms, in particular Bacteria, or eukaryotic cells, especially yeasts, used. The bacterial strain is particularly preferred Escherichia coli or the yeast strain Saccharomyces cerevisia.

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

Furthermore, at least one first becomes regulatory Factor or the corresponding gene arrangement provided. According to the present invention, the at least affects a first regulatory factor one or more second regulatory factors and not directly the activity of the  Reporter system, which makes it possible for the first time, the Activity of a first regulatory factor by a detect positive signal.

The at least one second regulatory factor is determined by encoded at least a second gene arrangement. Because of Simplification is in the already mentioned and in the following components involved in the process not always explicitly mentioned that both one and several components such as genes, gene arrangements, regulatory Factors, proteins, etc. can be involved. It should however, be designed to take advantage of these opportunities are included. The activity of the second is preferred regulatory factor through the first regulatory Factor influenced. About an interaction of the second regulatory factor with components of the reporter system will also affect the activity of the reporter system taken. Thus, according to the present invention, the Activation of the reporter system by adding a inhibitory component on the interaction of the first and second regulatory factors.

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

According to a further preferred embodiment of the inventive method contains the Transcription regulator at least two hybrid proteins,  in particular two hybrid proteins, which of one provided third gene arrangement are encoded.

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

The regulatory components of the first regulatory Factor are not up according to the present procedure regulatory proteins restricted. For example, the first regulatory factor nucleic acids or others Components included.

The activity of at least a 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 components of the first modified by mutagenesis regulatory factor.

In a further preferred embodiment, a fourth gene arrangement for the expression of peptides provided. These peptides have one in particular inhibitory activity. So in vivo synthesized peptide libraries for the inhibition of regulatory factors are used. In others preferred configurations are any combinations of the inhibitors mentioned.  

The action of the inhibitory is particularly important Components on the activity of the first regulatory Factor by influencing the interaction between at least two regulatory components in the first regulatory factor are included. This Action is preferably an inhibition of the interaction of the regulatory components. The activity can also be due several components, e.g. B. in a multiprotein complex, be regulated. This complex is active as long as single or multiple building blocks form this complex. First inhibiting one or more of these components destroys the activity of the entire complex. Also from The previously described deviating activities that occur in somehow the expression of the second regulatory Activate factor are the first regulatory factor suitable. The inhibition of the first regulatory factor can both the activity of the transcription regulator and / or the generation of the transcription regulator affect. In particular, the interaction between two regulatory proteins of the first regulatory factor inhibited. If one of the interactions mentioned is inhibited, lies an inactive or reduced in activity first regulatory factor. As a result, the Interaction of the first regulatory factor with the second regulatory factor inhibited or reduced. In Another preferred embodiment is the Interaction between the first regulatory factor and the second regulatory factor. This too Action is particularly preferably an inhibition. In a particularly advantageous embodiment affects the Inhibitor of the first regulatory interaction Factor with gene segments of the second gene arrangement. In a further embodiment of the method according to the invention modifies the at least one first regulatory factor, which preferably contains one or more proteins at least a second regulatory factor,  for example via kinization, dephosphorylation, Splitting, refolding, or changing conformation.

If no inhibitor is added, the first is regulatory Factor in particular in the active form and affects the Activity of the second regulatory factor. It is the further preferred that the first regulatory factor with DNA sections for the second regulatory factor encoding second gene arrangement interacts and thus the Expression of the second regulatory factor influenced.

The addition of an inhibitory component affects for example the interaction between at least two Components that together form the first regulatory Factor. In a further embodiment, the Addition of the inhibitory component to the activity of the first regulatory factor, regardless of whether it contains one or more regulatory components. The Influence on the activity affects, for example, the transcription-activating or binding activity of the first regulatory factor or the interaction of the first and second regulatory factor.

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

Addition of the inhibitory component is preferred the interaction between the first regulatory factor and a DNA portion of the second gene assembly that the encoded second regulatory factor, being in particular an inhibition. The Action or inhibition of the above Interactions through inhibitory components leads to an effect on the gene expression of the second  Gene arrangement, in particular to inhibit gene expression the second gene arrangement. The leads particularly preferably Addition of the inhibitory component via an inhibition of Activity of the regulatory protein to inhibit the Expression of the second gene arrangement.

According to a further preferred embodiment of the The inventive method is by adding a inhibitory component on the interaction between acted at least two components, one of these Component is a regulatory component or a contains regulatory component.

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

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

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

The second protein components are especially protein components that Have anchoring functions. This is preferably done Anchoring the interacting Protein components in the cytoplasm. Has proven to be beneficial also anchoring the interaction with each other standing proteins via the anchoring function of the second Protein component proven in the membrane.  

According to a further advantageous embodiment inhibition by adding an inhibitory component the interaction between the at least two components the release of 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 is particularly preferred transcription activating factor by the released inhibitory factor inhibited, causing an inhibition the expression of the second gene arrangement takes place.

In the previously mentioned and also in the following Embodiments contain the regulatory or interacting components sections that the regulatory or interacting functions. The corresponding protein segments can be one or more Include sections or domains. These sections or Domains can be found in different regions of a protein or are on different proteins.

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

It has proven to be particularly advantageous that the at least a first protein component is a section that with the at least one second protein component interacts with a section that interacts with a Transcription factor interacts as well as an inhibitory Section contains, only after inhibition of the interaction the bond between the at least two protein components the inhibitory second protein component die Expression of the second regulatory factor inhibits.

According to a further preferred embodiment, the at least a first regulatory component  Transcription regulator or transcription factor of the second regulatory factor after inhibition of the Interaction with the at least one second Protein component reduced in its activity or is inactivated, causing inhibition or reduction the activity of the second regulatory factor takes place.

The second regulatory factor is especially around proteins, for example, depending on Experimental arrangement to at least one repressor or at least one recombinase. The active second regulatory factors work through an interaction Components of the reporter system on the activity of the Reporter system. The second regulatory factor is binding preferably on DNA sections of the reporter system.

A repressor, encoded by the second gene arrangement, is a Example of a second regulatory factor. This Repressor affects the expression of at least one Reporter gene by targeting components of the Reporter system binds. According to a preferred embodiment the method of the invention, the binding of Repressors on components of the reporter system by others Agents regulated. For example, that induces Antibiotic tetracycline a replacement of a prokaryotic Repressors from DNA. An active repressor inhibits that Expression of at least one reporter gene. Against, for Example by inhibiting the interaction of the hybrid proteins of the transcription regulator the expression of the repressor Inhibited by a gene, at least one expression occurs Reporter gene.

A recombinase encoded by the second gene array is another example of a second regulatory Factor. Included in a corresponding experimental set-up the reporter systems recombination elements. There is an active one  first regulatory factor before, eliminated or inverted the recombinase via recombination processes at least one Reporter gene. The recombinase interacts with the specific, the reporter gene or the reporter genes flanking recombination elements and eliminated or inverts the reporter gene, which of the Recombination elements is flanked. Both after one Elimination as well as after inverting becomes the reporter gene not expressed. So is not an inhibitor of the experimental approach added, there is an active first regulatory Factor that prefers that for a second genes encoding regulatory factor activated, especially about interaction with sections of DNA. The second active factor, such as B. a repressor or Recombinase, inhibits the expression of the reporter genes.

By influencing the interaction of at least two regulatory components of the Transcription regulator, the regulatory Components, in particular two hybrid proteins, are described in a preferred embodiment of the invention The expression of at least one recombinase controlled, causing a change in expression at least of a reporter gene. 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 in particular inhibited it the at least one reporter gene is expressed.

An embodiment is particularly preferred in which the interaction on the interaction of at least two Protein components the expression of at least one repressor Gene is controlled, causing a change in expression of the at least one reporter gene.  

In particular, by inhibiting the interaction of the Protein components inhibited the expression of the repressor gene and expression of the at least one reporter gene he follows.

According to a further preferred embodiment of the invention is affected by the interaction of the at least two protein components expression at least a recombinase controlled, causing a change in Expression of at least one reporter gene takes place.

Inhibition of the interaction is particularly preferred Protein components inhibited the expression of the recombinase and the at least one reporter gene is expressed.

The tests are based on the fact that among the given Test conditions at least one gene product of the at least a reporter gene is detectable.

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

The detection of the gene product or products takes place then, for example, about one or more changes to the Phenotype of host cells. In a particularly preferred Embodiment enables the gene product of the reporter gene Cell growth of the host cells in deficient medium. The gene product e.g. B. the reporter gene Leu2 enables cell growth in leucine deficient medium. Another advantageous Modification of the method according to the invention are yeast strains with chromosomal mutations used as host cells for example on leucine deficits in the Metabolism of the amino acid leucine. The  Chromosomal mutations can also cause deficits in the Metabolism of the amino acids tryptophan and histidine to lead. If necessary, the use is also Protease-deficient yeast makes sense.

It is also preferred to provide gene arrays that code for one or more gene products, these Convert gene products to substrates in a measurable color reaction can, such as B. the reporter system LacZ. The gene product this reporter gene, the β-galactosidase, reacts with different substrates in a visible color reaction.

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

According to the present invention, therefore, by use one or more inhibitory components first the Influences the activity of the first regulatory factor, which in turn depends on the activity of the second regulatory factor acts.

The second regulatory factor ultimately affects that Expression of the reporter gene. According to the present invention by adding one or more inhibitory components the activity of the first regulatory factor inhibited, this is caused also inhibited the second regulatory factor, whereby according to a preferred embodiment, the second regulatory factor itself inhibited or in another preferred embodiment, the expression of the second regulatory factor is inhibited. Since in no case  active second regulatory factor, it will Reporter genes or the reporter genes are expressed. First the Inhibition of the first regulatory factor therefore brings about expression of reporter genes. The respective phenotype depends on whether the second regulatory factor is formed will or not. One should be particularly emphasized further embodiment of the present invention, wherein in one host organism two gene arrangements for two second regulatory factors, especially gene arrangements for a repressor and a recombinase, in addition to those in e.g. B. Claim 1 mentioned others for carrying out the experiment necessary gene arrangements are provided.

Depending on the test condition, one of the two becomes second regulatory factors, d. H. especially repressor or Recombinase, or both are expressed simultaneously. This results in an increased specificity of the method reached.

The inhibition of the activity of the transcription factor or of the transcription regulator or its generation finally to activate the reporter gene or 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 so. B. selectively detectable in growth experiments, the cells acted on by the inhibitor, 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 ⁹ different molecules). It is therefore possible for the first time with the method according to the invention to carry out an identification of cells after the addition of inhibitor, in particular also due to growth. This proves to be very advantageous if, for. B. 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. Furthermore, it is possible to make statements regarding the specific site of action of the inhibitor, since targeted at z. B. regulatory factors are acted on and the reporter genes are only expressed after inhibition of the factor. 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. The method can be used in such a way that the inhibiting substances and peptides are used for the development of therapeutic agents. In a particularly preferred embodiment, peptide libraries synthesized in vivo are provided to inhibit the first regulatory factor.

Preferred embodiments of the Illustrated method according to the present invention, the execution taking into account a active or inactive first regulatory factor is explained. It is assumed that the Proteins as regulatory factors.

As reporter gene z. B. the Leu2 gene, which growth on leucine-deficient medium, and the LacZ gene, whose gene product, the β-galactosidase, 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). Similarly to Leu2, others can Genes that encode enzymes from biosynthesis, such as. B. the URA3 gene, because of its expression correspondingly deficient yeast strains complement and thus Growth on Uracil can enable deficient media (Sherman et al., Laboratory Course Manual for Methods, In: Yeast Genetics, (1986)). Also the activity of luciferase or chloramphenicol acetyltransferase can easily and can be quickly detected in enzymatic reactions (Ibelgaufts, genetic engineering from A to Z (1990) VCH-Verlag (Weinheim)).

The invention is described below with reference to the accompanying Drawings explained in more detail.

Fig. 1a and 1b show gene arrangements schematically illustrated for a repressor as well as the reporter proteins, the interaction is shown an active (Fig. 1a) and an inactive (Fig. 1b) transcription factor with the DNA.

FIGS. 2a and 2b show gene arrangements schematically illustrated for a repressor as well as the reporter proteins, the interaction is shown an active (Fig. 2a) and an inactive (Fig. 2b), transcription regulators with the DNA.

Fig. 3a and 3b show gene arrangements schematically illustrated for a recombinase, and for reporter proteins, wherein the interaction is shown an active (Fig. 3a) and an inactive (Fig. 3b) transcription factor with the DNA.

FIGS. 4a and 4b schematically illustrate gene arrangements illustrated for a recombinase, and for reporter proteins, wherein the interaction of an active (Fig. 4a) and an inactive (Fig. 4b) transcriptional regulators is shown with the DNA.

Fig. 5a and 5b show gene arrangements schematically illustrated for a repressor as well as the reporter proteins, the interaction is shown an active (Fig. 5a) and an inactive (Fig. 5b) transcriptional regulators with the DNA.

Fig. 6a and 6b show gene arrangements schematically illustrated for a recombinase, and for reporter proteins, wherein the interaction is shown an active (Fig. 6a) and an inactive (Fig. 6b) transcriptional regulators with the DNA.

Fig. 7a and 7b show gene arrangements schematically illustrated for a repressor as well as the reporter proteins, the interaction is shown an active (Fig. 7a) and an inactive (Fig. 7b), transcription regulators with the DNA.

Fig. 8a and 8b show gene arrangements schematically illustrated for a recombinase, and for reporter proteins, wherein the interaction is shown an active (Fig. 8a) and an inactive (Fig. 8b), transcription regulators with the DNA.

Fig. 9a and 9b show gene arrangements schematically illustrated for a repressor LexA GST, wherein the interaction is shown an active (Fig. 9a) and an inactive (Fig. 9b) the first regulatory factor AD1-Tet having the DNA.

Fig. 10a and 10b show gene arrangements schematically illustrated for a second regulatory factor Tet-GST, wherein the interaction of an active (Fig. 10a) and an inactive (Fig. 10b) the first regulatory factor LexA CTF7 is represented by the DNA.

FIG. 11a and 11b schematically illustrate gene arrangements illustrated for a second regulatory factor Tet-GST, wherein the interaction of an active (Fig. 11a) and an inactive (Fig. 11B) the first regulatory factor LexA ctf2 - AD1- TIM shown with the DNA is.

The targets (targets) or transcription-stimulating targets mentioned in FIGS. 1 and 3 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.

The targets mentioned in FIGS. 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 being the regulatory component with the transcription activating activity.

In the case of in Figs. Targets 5 and 6. (targets) is the interacting components of the first regulatory factor, said target I-binding regulatory component of both the DNA and the transcription-activating activity and Target II is the component interacts with the target I and is only present in the active form in the case of undisturbed interaction.

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 Interaction of the target II solved 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 in particular Protein segments called the respective activity or effect the relevant function. Here, a or multiple domains. The domains or Sections of protein can be found in different areas Protein or localized on different proteins.

The binding points of these factors are accordingly Target binding sites designated.

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

Yeast plasmids

To express the different genes in the underlying assay will be both commercially available Plasmids (e.g. pYES2 from Invitrogen, the Netherlands), as also other constructs (Altmann, H. Dissertation (1994), Altmann H. et al., Proc. Natl. Acad. Sci., USA (1994) 91, p. 3901-3905). They usually have plasmids a place of replication to replicate their DNA in yeasts (eg "2 µm-ori") and one for replication in Bacteria (e.g. "colE1-Ori"). Furthermore, marker genes for the selection of these plasmids in the two organisms used (e.g. URA3, HIS3, TRP1 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. Gal1 promoter) or specifically constructed target-dependent promoters (NFI, Tet, LexA) respectively.

The individual elements can be cloned into plasmids instead of the assay into the genome of the yeast strain used be integrated (Sherman et al., Laboratory Course Manual for Methods, In: Yeast Genetics, (1986)). Such in the Items located in yeast do not need their own Origin of replication and selection marker.

The following elements are used in the test system:

• a) 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).
  Gal1 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) using the restriction enzyme SpeI.
  Inactive Gal1 / Gal10 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 Hindill, this promoter element is isolated from the vector pLR1Δ1 constructed in this way 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:
  Tet-dependent promoter: The 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 ₁ / O ₂ :
  LexA-dependent promoter: 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. Sci. 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 Gal1 promoter, this promoter is extracted from plasmid JK101 (Golemis et al., Mol. Cell. Biol. (1992) 12, p. 3006) using the BamHI restriction enzyme -3014) isolated and used for further cloning.
  loxP-dependent promoter: The Hoess et al. (Proc. Natl. Acad. Sci. USA (1982) 79, pp. 3398-3402) described loxP elements which enable the recombinase Cre of Coliphagen P1 to delete or invert sequences lying between two such elements are synthesized as an oligonucleotide and used for the construction of Cre-dependent promoters.
  Sequence for the loxP recombinase element:
  
• b) reporter genes
  LacZ gene: The LacZ gene is isolated via the restriction enzymes BamHI and Hindill 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:
  Tet gene: The Tet gene is isolated using the restriction enzymes XbaI 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 .
  Cre1 gene: The Crel gene is isolated by means of a PCR reaction from Coliphagen P1 (Hoess et al., J. Mol. Biol. (1985) 181, pp. 351-363) and used for the further cloning.
  Sequence primers used:
  
• 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 Hindill and EcoRI from the plasmid pEG202, a derivative of the vector LexA202 with an additional polylinker sequence behind the LexA gene (Ruden 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 ₁ (amino acid 521-551) of the NFkB protein is derived from the plasmid pLexTA ₁ using the restriction enzyme EcoRI (Schmitz et al., J. Biol. Chem. (1994) 269, S. 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 3 X from Pharmacia (Sweden) using restriction enzymes and used for further cloning.
  AD1-Tet: The fusion gene AD1-Tet is composed of the acidic activation domain AD1, isolated from the plasmid pJG4-5 (Gyuris et al., Cell (1983) 75, pp. 791-803) with the aid of the restriction enzymes HindIII and EcoRI, and the coding sequence for the Tet repressor (see above).
• d) 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:
  "Random oligo pool": To construct a "random oligo pool", a single-strand oligonucleotide pool of the following composition is synthesized:
  Sequence:
  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 procedure

The cultivation and transformation of bacteria, yeast and higher eukaryotic cells occur after 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, N.Y .; Sambrook et al., Molecular Cloning (1989), Cold Spring Harbor Laboratory Press; Lindl et al., (1987); Altmann et al., Proc. Natl. Acad. Sci. (1994) 91, p. 3901- 3905).

The bacterial strains JK101 from Stratagene (Germany) and Top10 from Invitrogen (Germany), as well as the Yeast strains INVSc1 and INVSc2 from ITC (Germany) were used for the experiments.  

Determination of β-galactosidase activity, the LacZ Gene product, takes place in so-called β-galactosidase assays (Altmann et al., Proc. Natl. Acad. Sci. (1994) 91, p. 3901- 3905).

For the determination of the CAT enzyme activity in protein extracts according to the immunoassay principle, for example, that of Company Boehringer (Germany) commercially available "CAT ELISA test used.

example 1

This example represents the configuration shown in FIG. 1.

• 1. A gene arrangement is provided which for encodes a transcription factor. This Transcription factor binds to DNA areas of a DNA (Target binding site) for a second regulatory factor, in this experimental setup for a repressor, coded.
• 2. Furthermore, a repressor arrangement is included a binding site on the DNA (target binding site) for the transcription factor mentioned under 1 provided.
• 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) preferably binds, one or more repressor recognition sequences to which a repressor can bind, and a TATA box for basal transcription together.
  • 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, ie there is no growth of the host organisms, and no color reaction can be detected.
  • 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. There is therefore no repressor 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. As a result, the yeasts grow in leucine deficient media, and after growth in media 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 one encoded another arrangement for a hybrid protein, which is a fusion protein of an activation domain of the transcription regulator and a second protein component ( Fig. 2a).
• 2. Furthermore, a repressor arrangement is included one or more binding sites on the DNA (target Binding site) for the transcription regulator provided.
• 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 codes for the repressor gene, and causes expression of the repressing 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. If 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 is inhibited. There is now no active transcription regulator or a transcription regulator whose activity is reduced, 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 or a change in color (blue color) 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.
  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 for a Transcription factor encoded is provided. This transcription factor binds to areas of one DNA (target binding site) for a recombinase coded.
• 2. Furthermore, a gene arrangement is provided, which for the sequence-specific recombinase of Coliphagen P1 coded.
• 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 an endogenous activator preferably 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, an active transcription factor is present. This transcription factor positively regulates the expression of the recombinase Cre. The recombinase interacts with the lox P sequences which flank the reporter genes LacZ / Leu2 and eliminates or inverts the reporter genes mentioned in a recombination process.
    Accordingly, no functional reporter genes are expressed, there is no growth of the host organisms, and there is also no detectable color reaction after growth on a medium containing X-Gal.
  • b) After the addition of 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 in Example 2 described, provided that for interactive Encode hybrid proteins that are Protein interaction an active transcription form regulator.
• 2. Furthermore, a gene arrangement is provided, which for the sequence-specific recombinase Cre des Coliphagen P1 coded. This recombinase corresponds 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 protein-protein interaction takes place between the fusion proteins forming the transcription factor, with which an active transcription factor is present. The active transcription factor binds to a binding site on the DNA, this DNA containing the cre gene coding for the Cre recombinase. After the transcription factor has been bound, 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, 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.
  • b) 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 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 use of a recombinase in the present method enables inhibitor detection according to an "all or nothing" principle, wherein, 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. 5.

• 1. Gene arrangements are provided which code for two interacting proteins, an 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 a further second protein which contains at least one domain which interacts with the first protein ( FIG. 5a).
• 2. Furthermore, a repressor arrangement is included one or more binding sites on the DNA (target Binding site) for the transcription regulator provided.  
• 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, there is an undisturbed protein-protein interaction (Target I - Target II) and there is an active transcription regulator. The active transcription regulator binds to its corresponding binding site on the DNA (target I binding site), which codes for the repressor gene, and causes expression of the repressing 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. B. 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 is 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).
    The DNA-binding domain of the one protein of the transcription regulator can bind or not bind to the relevant DNA section, 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 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 TF. 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 in Example 5 described, provided that for interactive Encode proteins, which have a protein-protein Interaction with an active transcriptional regulator form.
• 2. Furthermore, a gene arrangement is provided, which for the sequence-specific recombinase Cre des  Coliphagen P1 coded. This recombinase corresponds 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 5, if there is no inhibitor, there is an undisturbed protein-protein interaction between the proteins forming the transcription regulator, with which an active transcription regulator is present. The active transcription regulator binds to a binding site located on the DNA, this DNA containing the Cre gene coding for the Cre 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, 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.
  • b) After adding an inhibitor to the test mixture, the protein-protein interaction of the proteins, e.g. B. 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. It becomes a gene arrangement for one Transcription factor Target I provided the can be inhibited.
• 3. Furthermore, a repressor arrangement with one or more binding sites on the DNA (target Binding site) for the transcription factor provided.
• 4. A reporter gene arrangement as described under Example 1, point 3 is also provided.
  • a) In the case of 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 site 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.
  • b) If an inhibitor is added to the experimental set-up, e.g. B. 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.
    Due to the inhibited protein-protein interaction, there is no active transcriptional regulator in any case. 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 or a change in color (blue color) 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 8

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

• 1. There are gene arrangements as in Example 7 described, provided that for interactive Encode proteins.
• 2. It becomes a gene arrangement for one Transcription factor provided (Target I).
• 3. Furthermore, a gene arrangement is provided, which for the sequence-specific recombinase Cre des Coliphagen P1 coded. This recombinase corresponds 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, with which an active transcription factor is present. 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 the transcription factor has been bound, 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, 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.
  • b) 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. 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.

Example 9

The method of operation is illustrated using the example of Tet repressors and its specific inhibition by Tetracycline explained.

This example represents the configuration shown in FIG. 9.

• 1. A gene arrangement is provided, which for the encoded first regulatory factor AD1-Tet. This regulatory factor binds to DNA areas of a DNA (Tet binding site) for the second regulatory factor, the repressor LexA-GST, coded.
• 2. Furthermore, a repressor arrangement of the Repressors LexA-GST with a binding site on the DNA (Tet binding point) for the first mentioned under 1 regulatory factor AD1-Tet provided.
• 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) preferably binds, one or more LexA binding sites to which a LexA-GST can bind, and a TATA box for initiation of the basal transcription together.
  • a) Expression of the fusion clone AD1-Tet (first regulatory factor) stimulates the expression of the repressor LexA-GST (second regulatory factor). This repressor protein in turn, by binding to the LexA binding site of the LacZ or LEU2 promoter described in FIG. 9, represses 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 the reporter genes is inhibited by glucose and only stimulated to galactose, these yeasts cannot grow on leucine-deficient media and remain white on X-Gal-containing medium (Table 1). If the medium has galactose as a sugar source, the Gal1-LexA promoter remains inhibited by the AD1-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 ( FIG. 9 and Tables 1 and 2), the binding of the AD1-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 now no longer able to inhibit the activity of the reporter system. The yeast cells now turn blue with galactose as the sugar source and X-Gal as the substrate for the 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 media deficient in leucine and remain white on media plates containing X-Gal. Other antibiotics such as ampicillin, chloramphenicol, kanamycin and carbenicillin do not have such a repressing effect on the first regulatory factor AD1-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:

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

• a) Evidence of the functioning of the individual elements in the assay
  • 1. AD1-Tet as a transcription factor:
    In order to demonstrate that the fusion clone AD1-Tet (first regulatory factor) is functionally expressed in the yeast, transported into the nucleus and efficiently bound there to the DNA consensus sequence and from there can activate the transcription of a gene, the following assay was carried out :
    AD1-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 AD1-Tet protein, there is also no expression of the reporter gene LacZ. The colonies remain white.
  • 2. 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 AD1-Tet protein, increasing concentrations of the antibiotic were added to the media plates:

Table 3:

As the experimental data show, depending on the increasing tetracycline concentration (Table 3), the binding of the AD1-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 "++").

• 1. LexA-GST as a repressor
  In order to demonstrate that the second regulatory factor (LexA-GST) is functionally expressed in the yeast, transported into the nucleus and efficiently bound there to the LexA consensus sequence and from there can inhibit the transcription of a gene, the following assay was carried out.
  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 unable to recognize the LexA binding sites and cannot repress the activity of Gal4. The reporter gene LacZ is expressed and the colonies turn blue (Altmann et al., Proc. Natl. Acad. Sci. (1994) 91, 3901-3905).

Example 10 Identification of specific peptide inhibitors of CTF7

To screen inhibitory peptide sequences against yeast transcription-active CTF7, a member of the NFI family (Altmann et al., Proc. Natl. Acad. Sci. (1994) 91, 3901-3905), the following assay ( Fig. 10) performed.

This example represents the configuration shown in FIG. 10.

• 1. A gene arrangement is provided which for a first regulatory factor LexA-CTF7 coded. 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 areas of a DNA (LexA- Binding site) for a second regulatory Coded factor Tet-GST.
• 2. Furthermore, a gene arrangement for the second regulatory factor Tet-GST with a binding site on the DNA (LexA binding site) for the under 1 first regulatory factor called LexA-CTF7 provided.
• 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) preferably binds, a Tet binding site to which Tet-GST can bind and a TATA box for initiation the basal transcription together.
  • 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.
  • b) After the addition of 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 thus 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 media containing X-Gal, a color change (blue) occurs.
    According to the present invention, the addition of the Trx peptide thus leads to expression of the reporter gene (s) by the repressing or not repressing the second regulatory factor Tet-GST.
    The test is carried out in detail as follows:
    The yeast strain INVSc1 was transformed with the plasmids shown in FIG. 10 and the transformation mixture was spread on a minimal medium plate containing glucose (20 × 20 cm; uracil, tryptophan and histidine deficient for selection on the plasmids).
    The vector pEG-CTF7 (corresponds to the first regulatory factor) and was according to Altmann et al. (Proc. Natl. Acad. Sci. (1994) 91, 3901-3905).
    pYES-Leu2 / 34-TetGST (plasmid which encodes the second regulatory factor TetGST and contains the reporter system).
    The plasmid pYES2 served as the starting vector. The LexA binding sites oligos were ligated into the XhoI site of the inactive Gal1 / Gal10 promoter, the entire promoter was isolated with BamHI and cloned together with the fusion clone Tet-GST into the polylinker of the plasmid pYES2. The Gal1 promoter had previously been deleted via the SpeI interfaces. In the NheI 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 Gal / Tet promoter was ligated into the reconstituted BamHI interface. This was obtained by cloning Tet oligos and then ligating Gal4 binding sites into the XhoI site of the inactive Gal1 / 10 promoter.
    pYES2 (TRP1) TRX oligo pool (peptide pool express 08059 00070 552 001000280000000200012000285910794800040 0002019502584 00004 07940 of the construct; complexity approx. 10 ⁵ )
    By ligation of the Trp1 gene in the pYES2 vector linearized with ApaI and NheI, the Ura3 gene was destroyed and the plasmid was selectable for tryptophane deficiency. The Trx gene isolated via PCR was then cloned into the EcoRI, XhoI linearized vector. The pool fragments were ligated via the RsrII 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-TA ₁ different from CTF7 in yeasts. In this way, peptides that specifically inhibit CTF7 can be distinguished from non-specific inhibitions.

Table 4:

The inhibitors B and C also turned blue on plates containing glucose, that is to say irrespective 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 ₁ domain. The inhibitors A, E, F and G, however, were only able to repress the activity of CTF7 (nb: not determined).

Example 11

Identification of specific inhibitors of 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 INVSc1 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. Sci. (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 / AD1-TIM.
• 2. pYES-Leu2 / 34-TetGST (plasmid, which the second regulatory factor TetGST encoded and that Reporter system included).  
• 3. pYES2 (TRPI) TRX oligo pool (construct expressing peptide pool; complexity approx. 10 ⁵ ).
  • a) The first regulatory factor LexA-CTF2 / AD1-TIM is composed, as described above, of two fusion proteins LexA-CTF2 and AD1-TIM, an active first regulatory factor pSH only when the protein-protein interaction of the two fusion proteins is undisturbed -CTF2 / TIM is present (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 / AD1-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 reproter genes. 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. B. 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.
    Due to the inactive first regulatory factor LexA-CTF2 / AD1-TIM, no expression of the Tet-GST gene (repressor gene) takes place. Since there is no repressor Tet-GST, the reporter genes Leu2 and / or LacZ are expressed after binding a further transcription factor Gal4.
    The test is carried out in detail as follows:

The approx. 35,000 colonies were collected, 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. 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 (Lac2 and Leu2) and the transcription factor LexA-TA ₁ in yeasts. In this way the TIM-CTF2 interaction specifically inhibiting peptides could be separated from non-specific ones.

Table 5:

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 LexATA ₁ domain. In contrast, inhibitors B and F were not able to repress AD1-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 the inhibitory activity of various inhibitors, ie in addition to 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 amplifications are also possible and detectable in the expression of the reporter genes.

Claims (64)

1. A method for determining the activity of at least one first regulatory factor, which can be detected via the activity of at least one reporter system, 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,
• c) adding at least one component which has an inhibitory effect on the first regulatory factor,
• d) influence on the activity of the second regulatory factor by the at least one first regulatory factor,
• e) interaction of the second regulatory factor with components of the reporter system, thereby acting on the activity of the reporter system, and
• f) detection of the activation of the at least one reporter system by adding the at least one inhibitory component via the interaction of the first and second regulatory factors, an inhibition of the at least one first regulatory factor by the at least one inhibitory component leading to an activation of the at least one reporter system should.

2. The method according to claim 1, characterized in that that the first regulatory factor with DNA  Sections of regulatory for the second Factor-coding second gene arrangement interacts, whereby on the expression of the second regulatory factor is affected. 3. The method according to any one of claims 1 or 2, characterized characterized 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 a first regulatory factor one or more regulatory Contains 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 regulators Proteins one or more transcription regulators are. 7. The method according to claim 6, characterized in that the transcription regulator or the Transcription regulators one or more Are transcription factors. 8. The method according to any one of claims 5 to 7, characterized characterized in that the regulatory protein or regulating proteins the at least a second Modify regulatory factor. 9. The method according to any one of the preceding claims, characterized in that the modification of the at least a second regulatory factor  through kinization, dephosphorylation, cleavage, Refolding or change of conformation takes place. 10. The method according to any one of the preceding claims, characterized in that by adding the inhibitory component on the interaction between at least two components, which together form the first regulatory factor, and / or on the activity of at least one first regulatory factor. 11. The method according to claim 10, characterized in that by adding the inhibitory component the interaction between at least a first one regulatory factor and a DNA section of the at least one second gene arrangement acted becomes. 12. The method according to any one of the preceding claims, characterized in that influencing the mentioned activity of the first regulatory Factor due to inhibitory components Effect on gene expression of the second Gene arrangement leads. 13. The method according to any one of claims 5 to 12, characterized characterized that an inhibitor has an inhibition the activity of the regulatory protein to one Inhibition of expression of the second gene arrangement leads. 14. The method according to any one of claims 1 to 9, characterized characterized in that by the addition of the inhibitory component on the interaction is acted between at least two components,  wherein the at least one first component is a regulatory component is or contains. 15. The method according to claim 14, characterized in that the regulatory component is a Protein component is or contains. 16. The method according to claim 15, characterized in that the at least one first protein component Is fusion protein. 17. The method according to any one of claims 14 to 16, characterized characterized that the regulatory component is or contains an inhibitory component. 18. The method according to any one of claims 14 to 17, characterized characterized in that the at least one second Component is at least one protein component or contains. 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 characterized in that the at least one second Component has an anchoring function. 21. The method according to claim 20, characterized in that about an interaction of the at least two Protein components these protein components in Cytoplasm are localized. 22. The method according to claim 20, characterized in that that about an interaction of the at least two  Protein components these protein components on or are localized in a membrane. 23. The method according to any one of claims 14 to 22, characterized characterized in that by the addition of a inhibitory component via an inhibition of Interaction between the at least two Components the at least a first component, which contains the inhibitory section, is released and with the transcription-activating factor of the gene arrangement interacts for the second regulatory factor. 24. The method according to claim 23, characterized in that the transcription activating factor by the released inhibitory factor inhibited , thereby inhibiting the expression of the second Gene arrangement takes place. 25. The method according to any one of the preceding claims, characterized in that as host organisms Microorganisms, preferably bacteria, or eukaryotic cells, preferably yeasts, are used become. 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 by an or  several changes in the phenotype of the Host organism occurs. 29. The method according to claim 28, characterized in that that the at least one gene product of the reporter gene Cell growth of host organisms in deficient medium enables. 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 substrates in can implement a measurable color reaction. 32. The method according to any one of the preceding claims, characterized in that the above Gene arrays arranged on different vectors are. 33. The method according to any one of claims 1 to 31, characterized characterized in that the gene arrangements mentioned are arranged in the same vector. 34. The method according to any one of claims 32 or 33, characterized in that the vectors are plasmids are. 35. The method according to any one of claims 32 to 34, characterized characterized in that one or more vectors with one or more gene arrays in the host genome are integrated.   36. The method according to any one of the preceding claims, characterized in that the second gene assembly encoded for at least one repressor. 37. The method according to claim 36, characterized in that that the repressor on the expression of at least acts on a reporter gene. 38. The method according to any one of claims 36 or 37, characterized in that the repressor by Binding to components of the reporter system works. 39. The method according to claim 38, characterized in that the binding of the repressor to components of the Reporter system regulated by additional agents can be. 40. The method according to any one of claims 36 to 39, characterized characterized in that in a reporter plasmid a LacZ Leu2 gene assembly and the repressor gene assembly are integrated. 41. The method according to any one of claims 1 to 35, characterized characterized in that the second gene arrangement for encoded at least one recombinase and the Reporter systems contain recombination elements. 42. The method according to claim 41, characterized in that that the recombinase via recombination processes eliminated at least one reporter gene or inverted. 43. The method according to claim 41 or 42, characterized in that the recombinase the sequence-specific recombinase Cre des Coliphagen P1  and is flanking at least one reporter gene 1ox 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 at least two Contains hybrid proteins, the hybrid proteins of a third gene arrangement can be encoded. 45. The method according to claim 44, characterized in that that a first hybrid protein is a fusion protein a DNA binding domain and a first Protein component and a second hybrid protein Fusion product from an activation domain of the Transcription regulator and a second Protein component is, by binding between the two protein components the active one Transcription regulator arises. 46. The method according to claim 45, characterized in that an impact on the interaction of the Hybrid proteins via inhibitory components he follows. 47. The method according to any one of claims 15 to 24, characterized characterized in that the at least two Protein components from a fifth gene assembly be encoded. 48. The method according to any one of claims 14 to 24, characterized characterized in that the at least one first Protein component a section that matches the at least a second protein component interacts with a section that interacts with a Transcription factor interacts as well as one contains inhibitory section, whereby only after  Inhibition of the interaction of the bond between the at least two protein components which are inhibitory acting first protein component the expression of at least a second regulatory factor inhibits. 49. The method according to any one of claims 14 to 16, characterized characterized in that the at least one first regulatory component a transcriptional regulator or transcription factor of the gene arrangement of the second regulatory factor is after Inhibiting interaction with the least a second protein component in its activity is reduced or inactivated, whereby the Expression of the second regulatory factor is inhibited. 50. The method according to any one of the preceding claims, characterized in that these inhibitory Components of natural products such as peptides, nucleic acids and carbohydrates or other chemical substances are. 51. The method according to claim 50, characterized in that the inhibitory components by mutagenesis changed 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 for the expression of peptides provided. 53. The method according to any one of claims 44 to 46 and 50 to 52, characterized in that by the Influence on the interaction of at least two  Hybrid proteins of the transcription regulator Expression controlled at least one repressor gene , which causes a change in the expression of the at least one reporter gene occurs. 54. The method according to claim 53, characterized in that that by inhibiting the interaction of the Hybrid proteins of the transcription regulator Expression of the repressor gene is inhibited and a Expression of the at least one reporter gene takes place. 55. The method according to any one of claims 44 to 46 and 50 to 52, characterized in that by the Influence on the interaction of at least two Hybrid proteins of the transcription regulator Expression controlled at least one recombinase is causing a change in expression at least one reporter gene occurs. 56. The method according to claim 55, characterized in that by inhibiting the interaction of the Hybrid proteins of the transcription regulator Expression of the recombinase is inhibited and a Expression of the at least one reporter gene takes place. 57. 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 at least two Protein components express at least one Repressor gene is controlled, creating a Change in expression of at least one Reporter gene is done. 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 expression of at least one Reporter gene is done. 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 at least two Protein components express at least one Recombinase is controlled, causing a change 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 expression of at least one Reporter gene is done. 61. The method according to any one of claims 41 to 60, characterized characterized in that in a host organism at the same time gene arrangements for at least one Repressor and at least one recombinase to be provided. 62. The method according to claim 61, characterized in that depending on the test conditions set Repressor or the recombinase or both expressed become. 63. Use of one of the methods according to one of the Claims 1 to 62 for the determination of inhibitory Substances preferred as lead structures can be used. 64. Use according to claim 63, characterized in that that the inhibiting substances are peptides.