EP1402057A2

EP1402057A2 - Method for detecting mutagenic substances

Info

Publication number: EP1402057A2
Application number: EP02735423A
Authority: EP
Inventors: Georg Reifferscheid; Claudia Schmid
Original assignee: Merck Patent GmbH
Current assignee: Merck Patent GmbH
Priority date: 2001-07-04
Filing date: 2002-06-25
Publication date: 2004-03-31
Also published as: AU2002310769A1; DE10132280A1; WO2003004697A2; WO2003004697A3; JP2004533260A; US20050070006A1

Abstract

The invention relates to a method and means for the rapid identification of mutations. The test strains used in the invention contain a reporter system and a selection marker. An antibiotic resistance protein, which after mutation allows the selective growth of the revertant and the targeted induction of the inducible reporter system of the developing revertants acts as the selection marker. Proteins that are capable of directly or indirectly triggering a measuring signal are used as the reporters. The mutations are identified by means of the reporter signal of the developing revertants, in such a way that the evaluation can take place after only a few hours.

Description

Method for the detection of mutagenic substances

The invention relates to a method and means for the rapid detection of mutagenic substances.

Bacterial mutagenicity and genotoxicity tests play a crucial role today as a screening process in product development in the chemical and pharmaceutical industries and in the routine analysis of environmental samples (industrial waste water,

"10 surface waters, room air condensates, sediments, suspended matter,

Soil samples etc.). The most commonly used mutation assay is the Arnes (Salmonella / microsome) mutagenicity test, which is also part of approval procedures or toxicology tests (Arnes, B.N., Lee, F.D. and Durston, W.E .; Proc. Natl. Acad. Sei. USA 70

- _j 5 (1973) pp. 782-786; OECD guidelines for the testing of chemicals). Other known genotoxicity tests are, for example, the umu test, the microplate version of which has found its way into standardization according to DIN (DIN 38415-3) and ISO (Reifferscheid, G., Heil, J., Oda, Y. and Zahn, RK, Mutation Res 253 (1991) pp. 215-222), the SOS chromotest

20 (Quillardet, P., Hofnüng, M., Mutation Res. 147 (1985) 65-78) and der

VITOTOX ^® assay (van der Lelie D, Regnier, L, Borremans, B., Provoost, A., Verschaeve, L, Mutation Res. 389 (1997) 279-90).

Umu-Tesi, SOS-Chromotest and Vitotox ^® -Assay detect changes in the molecular structure of the DNA of the test organism Salmonella typhimurium or Escherichia co // caused by genotoxins. The tests are based on the measurement of the genotoxicity-induced induction of genes of the bacterial SOS regulon. These genes are, for example, in an error-prone DNA repair system (SOS repair; umuDC genes in the umu test), in the inhibition of cell division involved in the SOS chromotest) and in repair and recombination (recN in the Vitotox ^® assay). Evidence is provided by photometric, luminometric or fluorometric measurement performed. The detection method depends on the type of (often plasmid-localized) reporter system (e.g. galactosidase, luciferase) which is linked to the respective SOS gene.

The umu test is carried out in state authorities concerned with environmental analysis and in

Companies in the chemical-pharmaceutical industry used for environmental monitoring and substance testing. As indicator tests for genotoxicity, umu-Test, SOS-Chromotest and Vitotox ^® -Assay record a broad spectrum of primary DNA damage, but they do not provide any direct evidence for mutations. in the

In contrast to mutations, primary DNA damage can still be repaired correctly and is then of no importance for the affected organism.

Another occasionally used genotoxicity test is the Mutatox ^®

Test (Bulich-A, publication number Ver. Wasser. Boden. Lufthyg. 89 (1992) 763-70). This test method detects genotoxicity in dark mutants of the luminescent salt water bacterium Vibrio fisheri. The exact mechanism of regaining luminescence after treatment with genotoxins has not been elucidated. It is believed that the

Reversion of the luminescence by various mutations, but above all by gene induction (similar to the SOS tests umu test, SOS chromotest and Vitotox ^® assay) or DNA intercalation (Bulich and Ulitzur: The mode of action of genotoxic agents in the Mutatox ^® test system, Microbics Corp., unpublished

Newsletters; Arfsten, DP, Davenport, R., Schaeffer, DJ. Biomed. Environ. Be. 7 (1994) 144-9). Since the test is only used occasionally, there is still no information about the correlation to mutagenicity tests such as the Arnes test. Contrary to the assumption of the detection of mutations implied by the name 'Mutatox ^® ', this test procedure is like all other SOS tests an indicator test. Strains of Salmonella typhimurium are used in the Ames test, which have lost the ability to grow on histidine-free culture media (agar plates with minimal nutrient medium) due to mutations in the histidine operon (His operon). The number of prototrophic His revertants which are formed under the influence of genotoxic substances by mutations in the His operon serves as a measure of the mutagenicity of the examined sample. The conventional Ames test is carried out with several strains of different mutagenic specificity (eg TA98, TA100, TA1535, TA97) as a plate incorporation assay [Maron, DM and Arnes, BN; Mutation Res. 113 (3-4), 1983, 173-215]. The sample to be tested is placed on the agar together with soft agar, a buffer or S9 mix for the metabolic activation of non-mutagenic substances and the test bacteria. Since the test substances diffuse more or less quickly into the agar during the incubation period (48 hours) depending on their solubility, an exact concentration of the sample to be tested, which affects the bacteria, cannot be determined. This type of application is therefore called a dose (eg 100 μg / plate).

The Amesll ™ test converted to liquid culture and carried out as a fluctuation assay in 384-well microtiter plates [Gee, P., Maron, D.M. and Arnes, B.N .; Proc.Natl.Acad.Sci.USA 91, 1994, 11606-11610] represents a further development of the conventional Arnes test, which now enables concentration-related studies. The growth of His revertants causes acidification and is indicated by the color change of a pH-dependent indicator in the selection medium. All wells that are positive with regard to the color change are counted without taking the color intensity into account. In addition to the grid thrust strain TA98, six strains (TA7001-

7006), specifically for the six possible base substitution types. In both test formats, the Ames test offers only a few automation options for routine operation. A prerequisite for partial automation of the Amesll ™ test is special laboratory equipment for inoculating and evaluating the 384-well microtiter plates.

Both the growth of the revertants into countable colonies (conventional Ames test) and the metabolic pH reduction (Amesll ™) require a 48-hour incubation phase, which is responsible for the long test duration. The selection of the

Revertants about nutrient deficiencies also run the risk of false positives when studying environmental mixtures that may be contaminated with nutrients. The long incubation period and the type of selection still require a high degree of sterility during the test in order to

Avoid growth of non-test-specific bacteria or fungi.

Due to the high expenditure of time, labor and materials, the use of the Ames test as a screening method with a large sample run is limited. The position of the target gene within the chromosome also makes it difficult to specifically examine the mutation. The target gene for the mutation is part of a large operon consisting of several genes, so that the actual reversion event can be overlaid by mutations in the other genes.

The object of the present invention was to develop a mutation test which combines the positive properties of the rapid indicator tests and the sensitive Ames test. According to the known indicator tests, it should be characterized by a quick and automatable implementation and should therefore be suitable for high-throughput screening (HTP screening). It was found that a specially modified test strain from the Enterobacteriaceaen group can be used to carry out a rapid and sensitive mutation test. The strains carry a reporter system and a selection marker switched off by targeted mutagenesis. An antibiotic is preferably used as the selection marker.

Resistance gene that enables selective revertant growth after reverse mutation. Proteins are used as reporters, which are preferably formed in proportion to the number of cells. Only mutant, growing cells can express the stringently controlled reporter after induction. The measurement is carried out directly or indirectly via a color reaction, a luminescence signal, a fluorescence signal or an electrochemical signal. With this special combination, different mutations in the selection marker can be detected indirectly via the reporter system formed only by growing cells. It is also possible to directly demonstrate the functionality of the target gene which has been regained through mutation if a selection marker with the properties of a reporter system is used. The actual mutagenicity detection is not carried out via cell growth as in the prior art, but via the expression of a reporter system, so that the evaluation can take place after only a few hours.

The present invention therefore relates to isolated prokaryotic test strains for use in a mutagenicity test, which have the following features: a) a reversibly mutated selection marker in the form of a resistance gene against bacteriotoxic or bacteriostatic substances or influences, b) a reporter system which leads directly or indirectly to a measurable Leads signal and can be detected selectively in reverted bacteria. In one embodiment, the selection marker can also serve as a reporter system. For example, the selection marker and reporter system can be identical.

In a preferred embodiment, the

Selection marker for an antibiotic resistance gene against an antibiotic that has a bacteriolytic effect or inhibits protein biosynthesis.

The selection marker is particularly preferably a tetracycline resistance gene, an ampicillin resistance gene, a kanamycin resistance gene, a chloramphenicol resistance gene or a streptomycin resistance gene.

in a preferred embodiment, the reporter system, when expressed, leads directly or indirectly to a color reaction, a luminescence or fluorescence signal.

In a preferred embodiment, the β-galactosidase, luciferase or β-lactamase gene or operon are used as reporter systems.

Also preferred are test strains which additionally have one or more of the following properties:. a cell wall permeable to large lipophilic molecules,

- a defective excision repair,

- A functional regulation unit of the SOS system

- the mutator genes umuDC and / or mucAB

The present invention also relates to a method for the detection of mutagenic agents, essentially characterized by the following method steps: a) providing at least one test strain according to the invention; b) incubation of the test strains from step a) with the potential mutagen; c) selection of revertants capable of growth; d) optionally induction of the reporter system (not applicable if the

Reporter system is at the same time a selection marker or if only a proof of growth control is to be carried out); e) direct or indirect detection of the gene product of the

Reporter system and / or proof of the growth of the reverted strains.

In step a), the provision of at least one test strain means that one or more test strains or a mixture of at least two test strains are provided.

In one embodiment, the selection marker serves as reporter system in step e), so that the detection of the gene product of the selection marker is carried out as detection of the gene product of the reporter system.

In a preferred embodiment, the growth is detected in step e) by means of a pH indicator in the medium.

The present invention also relates to a test kit for carrying out mutation tests, which contains at least one test strain according to the invention.

The present invention also relates to the use of at least one test strain according to the invention for HTP screening. Figure 1 shows the gene map of the plasmid of a test strain that can be used for the mutation test according to the invention.

Test strains which can be used for the mutation test according to the invention preferably contain a localized plasmid

Selection marker, the functionality of which has been reversibly switched off by targeted mutagenesis, and an inducible reporter system if the selection marker is not also a reporter system.

Bacteria are used as test strains. Prokaryotic test strains from the group of Enterobacteriaceae are preferably used, particularly preferably Escherichia coli or Salmonella typhimurium with a combination of specific properties defined in the genotype. The strains Salmonella typhimurium TA1535 (rfa, AuvrB) (McCann, J., Choi, E.,

Yamasaki, E. and Arnes, B.E .; Proc. Natl. Acad. Be. USA 72 (1975), pp. 5135-5139) or Escherichia coli EE122 (/ fa-like mutation, AuvrB) (Eisenstadt, E., Warren, AJ, Potter, J., Atkins, D. and Miller, H .; Proc. Natl. Acad. Sci. USA 79, 1982, 1945-1949). The strain EE122 is e.g. due to robustness and an active chromosomal u uDC operon.

A large number of strains are known from the prior art which carry a selection marker and an inducible reporter system. In contrast to these organisms, however, the selection marker of the test strains according to the invention has been mutated reversibly with loss of function. Only through the introduction of such a mutated one

Selection markers can be a test strain for the invention

Procedures are used. After treatment of the test strain with mutagenic agents, the reuptake of growth in the selection medium as a result of a reversion event in the target gene which codes for the selection marker is preferably determined quantitatively by the reporter system.

The reporter system of the test strains according to the invention is preferably inducible and is preferably formed in proportion to the number of cells present. The non-induced basal level should be as low as possible. The reporter system can be a single gene or an operon.

Genetic products that can be directly or indirectly detected photometrically, fluorimetrically, luminometrically or electrochemically can be used as reporters. Preferred gene products are GFP (green fluorescent protein), ß-lactamase, luciferase and particularly preferably ß-galactosidase, which can be detected in various known ways.

The advantage of the GFP is the possibility of detection without the addition of substrate. An influence on the activity of the reporter by inhibitory ingredients of the test material can thus largely be excluded.

If the selection marker and reporter system are identical, a direct detection of the mutation is possible. One example is the ß-lactamase, which mediates ampicillin resistance. The activity of the ampicillin gene restored by reverse mutation can be directly, for example, by using the substrate nitrocefin (O'Callaghan, CH, Morris, A., Kirkby, SK and Shingler, AH, Antimicrob. Agents Chemother. 1 (1972) 282-288 ; Sutton, LD, Biedenbach, DJ., Yen, A. and Jones, RN, Diagn. Microbiol. Infect. Dis. 21 (1995) 1-8).

On the other hand, the use of β-galactosidase can lead to a - caused by accumulation of the product of the enzyme reaction -

Amplifier effect can be exploited. The alternative luminometric measurement method also switches certain interference factors such as strongly colored test substances.

Reporter system and / or selection marker can be contained on the chromosome or on a stringently or relaxed controlled plasmid with a constant number of copies. The preferred number of Iow copy plasmids is 15-20 copies. The uniform distribution among the daughter cells should be ensured.

The reporter system is preferably under the control of a strictly regulated, inducible promoter. Such promoters are known to the person skilled in the art. The feM promoter is preferably used, which is repressed by the teπ ^ -coded repressor protein and, by adding tetracycline or a tet derivative, eg anhydrotetracycline, enables targeted induction of the reporter after the mutation has manifested. For an optimal induction of the in resistant (back-mutated) bacterial cells, atypical tetracyclines such as the anhydrotetracycline are preferred. The anhydrotetracycline derivative has a MIC ('Minimal Inhibition Concentration') value of 2 μg / ml im

Compared to 0.5 μg / ml for tetracycline less antibiotic activity [Olivia, B., Gordon, G., McNicholas, P., Ellestad, G. and Chopra, I Antimicrob. Agents chemother. 36, 1992, 913-919] and at the same time binds approximately 35 times more strongly to the Tet repressor [Degenkolb, J., Takahashi, M., Ellestad, GA and Hillen, W. Antimicrob. Agents chemother. 35, 1991, 1591-1595]. By inducing the β-galactosidase under the fetA promoter control with anhydrotetracycline, stronger signal values are achieved even at very low, non-inhibitory antibiotic concentrations.

A mutation-inactivated resistance gene is used as the selection marker in the test strains according to the invention, which allows the selective growth of the revertants after reverse mutation. Resistance proteins that allow targeted growth under selective conditions and whose coding gene can be reversibly switched off by mutation can generally be considered as gene products of the selection markers. The resistance mediated by the (intact) resistance gene can be a resistance to a bacteriotoxic or bacteriostatic substance such as an antibiotic or an action or influence (noxa, e.g. temperature or radiation). The resistance gene according to the invention is preferably an antibiotic resistance gene. Resistance genes against antibiotics which inhibit protein biosynthesis or have a bacteriolytic effect are particularly preferably used as selection markers. This special type of selection marker not only stops the non-mutated and therefore sensitive bacteria in the selection medium containing the antibiotic from growing, but also switches off protein biosynthesis. As a result, the signal background from non-mutated bacteria remains low after induction. It must be ensured that the selection pressure is maintained over the test period.

Standard media are used as the selection medium for the test strains and, if the selection is not caused by effects such as radiation or temperature, contain additives of the corresponding selection agent. The person skilled in the art is able, depending on the test strain used, the respective resistance mechanism and the number of resistance genes, to be suitable Compile selection media. The concentrations of the antibiotics in the selection medium are in the usual range. In the case of ampicillin, approximately 50 μg / ml are preferably used. The concentrations of the antibiotics depend on the type of plasmid on which the resistance gene is located, on the type of

Resistance mechanism and the test strain used.

Due to the antibiotic resistance used for the selection according to the invention, the selection of the medium has fewer limits than e.g. in the prior art when carrying out conventional mutagenicity tests. Such tests are based e.g. on the formation of prototrophic His revertants. This means that only minimal media can be used to ensure that the selection medium does not contain histidine. In contrast, any type of medium can be used for the method according to the invention which contains all the constituents which are necessary for the growth of the type of germ used. The selection is not made via a missing nutrient component, but preferably through the addition of an antibiotic. Therefore, full media are preferably used according to the invention instead of a minimal medium. This leads to better and faster growth of the reverted germs and thus enables the test to be carried out and evaluated particularly quickly.

The gene serving as a selection marker can have a certain type of mutation to detect a type of mutation. Furthermore, different mutation types of the target gene on a plasmid can cover different mutation types with only one test strain, provided that the spontaneous mutation rates of the combined mutation types are of the same order of magnitude. This leads to a significant reduction in labor and material costs, since the

Use of many strains and complex parallel determinations are no longer necessary. In the production of the test strains according to the invention by targeted mutagenesis [Kunkel, TA; Proc. Acad. Be. UNITED STATES; 82 (2) 488-92], using the PCR (polymerase chain reaction) technique and other DNA-modifying enzymes, the different mutation types

(Raster shift and base substitution mutations) were introduced directly into known or artificially generated regions with particular sensitivity to mutagenic events (hot spot regions) or into active centers of the antibiotic resistance genes during the synthesis of the plasmid concerned by means of a mutant pair of primers. The various types of mutations and suitable hot spot regions are known to those skilled in the field of mutation tests. Repetitive base sequences with a high guanine content or alternating GC boxes are particularly susceptible. G-A transitions and G-T transversions are common in base substitutions.

The stage of antibiotic resistance is to be regained by a reverse mutation in the sequence section changed by deletion, insertion or the exchange of bases. When changing the sequence sections that are responsible for the formation of the active center, it should be taken into account that usually only exact reverse mutations can completely restore the functionality of the target gene. If the mutation hot spot is located at the beginning of the reading frame of the target gene, a longer gene area is available for a back mutation. In the case of a base exchange, either an amino acid important for the functionality of the protein is preferably changed or a stop codon is introduced. This is then preferably before the gene segment that is important for the functionality of the protein. When introducing grid shear mutations, the possibility of opening alternative reading frames must be taken into account. In addition to the selection marker and reporter system, the test strains preferably have further properties which they have for the make the test more sensitive and universally applicable. This is primarily a cell wall that is permeable to large, lipophilic molecules (eg rfa or rfa-like mutation) and / or a defective excision repair (eg mutation in the uvrA or ß gene).

In addition, the test bacteria should preferably have a functional regulation unit of the SOS system. Different genes of the SOS system are very advantageous for a sensitive and complete detection of mutagenic substances. These include the genes recA, lexA, umuDC and / or mucAB. The homologous gene products UmuDC and MucAB are also referred to as mutator genes, because they increase or enable the formation of mutations in the bacteria in the course of genotoxic events. While umuDC is operably located on the E.coli chromosome, the muc \ ß-operon was isolated from naturally occurring plasmids (McCann, J. and Arnes, BE; Proc. Natl. Acad. Sei. USA 73 (1976) S. 950-954). These gene products often act synergistically and prevent an otherwise lethal stop of replication. The gene products RecA and LexA are used to detect DNA damage and regulate the induction of the genes of the SOS system.

Another advantage is the lack of antibiotic resistance in the parent strain.

When using test bacteria that do not have a chromosomal Umu operon, this or, preferably, the Muc operon can additionally be introduced via a plasmid. As a source for the sequence of the Muc operon, the plasmid pKM101 [McCann, J., Spingarn, NE, Kobori, J. and Arnes, BN; Proc. Natl. Acad. Be. USA 72 (3), 1975, 979-983], which can be isolated from the strain Salmonella typhimurium TA100 or TA98. After amplification by means of PCR [Mullis, KB and Falaona, FA; Methods Enzymol. 155, 1987, 335-350] and cloning can affect the operability of the Muc operon on the new one Plasmid to be checked in the Ames test. With the test substance nitrofurazone, which exerts its mutagenic effect mainly via the SOS system [Bryant and McCalla; Chem. Biol. Interact. 31 (2) 1980, 151-66], a significantly increased mutation rate compared to the control should be observed in the strains with the cloned Muc operon [Zeiger, E., Anderson, B., Haworth, S., Lawlor, T. , Mortelmans, K. and Speck, W; Environm. Mutagenic. 9 (9) 1987, 1-110]. This plasmid introduced by transformation methods must contain an origin of replication suitable for propagation in the test strain and a marker for the maintenance of the plasmid during growth. The mutated target gene and the inducible reporter system can also be located on this plasmid.

The production of several plasmids, each of which has only one special mutation, enables the mutagens to be differentiated, so that a mutation spectrum can be created. The mutation analysis using molecular biological methods is facilitated by the preferred position of the mutated gene on the plasmid. Furthermore, it is possible to detect several possible mutation variants simultaneously on one plasmid by amplifying the mutated gene serving as a selection marker with different types of mutations.

The cytotoxicity of the test material can be monitored by simultaneous use of a test strain which is analogous in genotype but not revertable.

The test strains used up to now (Ames test) are based on the recovery of prototrophic growth and are therefore susceptible to growth-promoting substances, which can have a negative effect especially when examining environmental samples with a high organic content. The selection according to the invention of the revertants, preferably via antibiotic resistance, can result in incorrect generation positive results are drastically reduced and contamination with non-test-specific microorganisms is avoided.

The present invention therefore also relates to a test kit for the rapid detection of mutagenic substances. This test kit for

Carrying out mutation tests comprises at least one or more test strains according to the invention, which can also be contained and / or used as a master mixture. Furthermore, optional components, such as the substrate of the reporter system, a stop solution or special selection or selection / indicator media can additionally be contained in the test kit. These additives are typically reagents that can also be purchased independently of the test kit. In order to make it easier for users to carry out the test using the method according to the invention, the test kit preferably additionally contains test instructions and a

Evaluation. The test kits according to the invention can be used for the individual detection of mutagenic substances or for HTP screening.

Figure 1 shows the example of a plasmid of a test strain according to the invention. The test strain contains a single copy plasmid or an Iow copy plasmid with a medium copy number (15-20 copies). Components of the plasmid are:

- an origin of replication, which guarantees a single plasmid or a medium number of copies (e.g. pMB1-Origin)

- The par region, which ensures an even distribution of the plasmid on the daughter cells

- the mutated selection gene as a target gene for mutations (amp ^s )

- the constitutively expressed repressor gene for the Tet repressor protein (tetR)

- the muc \ ß operon

- a resistance gene to maintain the plasmid (R) - The / acZ reporter system under the control of the tetA promoter

To carry out the method according to the invention, one or more test strains according to the invention are provided in a cell number of typically 10 ⁷ to 10 ⁹ / ml. Suitable media for the

Culture of bacteria is known to the person skilled in the art. A complete medium such as e.g. Luria-Bertani (LB-) medium used.

The test can be carried out in microtiter plates or similar vessels suitable for bacterial culture.

First the substance to be examined or the mixture of substances to be examined and, if appropriate, further reagents, such as e.g. S9 mix added. The cells are then pre-incubated for a certain period of time. Depending on the mode of action, the selection agent is added immediately or at a certain time during the preincubation. Pre-incubation is particularly necessary so that the cells can come into contact with the substances to be tested for a sufficiently long period of time so that damage that has been set can manifest itself as a mutation during replication. The pre-incubation is typically 0.5 to 2 hours.

The cells are then incubated in the selection phase for a period of typically 5 to 24 hours. The incubation time depends, among other things, on the pre-incubation time, the incubation temperature, the type of selection, the reporter system, the medium and the pre-dilution of the test batches. At 37 ° C typically 5 to 8 hours are needed for incubation, at 28-30 ° C typically 15 to 20 hours are needed. The reporter system is then induced by adding the appropriate induction agent and the mixture is typically incubated at 37 ° C. for 1 to 2 hours. If the reporter system is identical to the selection marker, this induction step is omitted. The mutagenicity of the substance to be investigated is determined by detecting the gene product of the reporter system. Depending on the reporter system, a photometric, fluorimetric, luminometric or electrochemical measurement is carried out.

Instead of or in addition to the detection of the revertants by induction of the reporter gene, they can also be detected via their growth. Especially when using a full medium, the growth of the revertants usually takes place so quickly that the detection can take place after about 16-18 hours. Evidence of

Growth can take place either via direct turbidity measurement or an indicator dye present in the medium. When the pH of the medium changes due to the growth of bacteria, the indicator changes color. Suitable indicators should show a color change between pH 7 and pH 5. Examples of suitable indicators are e.g. Bromocresol red or bromothymol blue. The indicator must have no genotoxic potential and should not have a decisive influence on bacterial growth.

With the help of this additional possibility of proof reverted

Germs, the user has two simple and sensitive detection systems at their disposal. The detection of cell growth is very simple and does not require induction of the reporter gene. The evaluation can be carried out by detecting the color change. This detection method only leads to qualitative results. A quantitative statement is possible if aliquots of the test batch are distributed over several batches (eg wells) and the number of batches that show a color change is related to the respective concentration of the test material. The preferred detection of the induction of the reporter gene, however, allows a direct quantitative statement, since the intensity of the signal can be determined. The method according to the invention has the advantage of being able to detect mutations very quickly. The substantial shortening of the incubation time compared to previous test systems and the selection method according to the invention enable extensive test execution automation. So far, only 1 to 2 test days per week could be carried out, but it is now possible to carry out 4 to 5 test cycles per week.

When using luminometric measurement methods, only a few mutated cells can be detected within a very short time, so that the method according to the invention provides a mutation test that can be carried out in a few hours, both for substance screening in industry and for testing environmentally relevant substances or multicomponent mixtures.

Another advantage of the method according to the invention is the insensitivity of the test strains used to mutations in metabolically relevant genes. In mutation tests in which minimal media have to be used due to the selection process, mutations in other genes relevant for metabolism and nutrient supply often lead to a significantly reduced growth of the revertants or even a death of the germs. The germs according to the invention, on the other hand, can be selected in full media, so that the germs can obtain many nutrients from the medium and react much less sensitively to mutations in metabolically relevant genes.

The method according to the invention is particularly suitable for use in product development in the chemical, pharmaceutical and cosmetic industries and in environmental analysis (waste water,

Surface water, sediments, suspended matter, ambient air, contaminated sites, pure substances). Even without further explanations, it is assumed that a person skilled in the art can use the above description in the broadest scope. The preferred embodiments and examples are therefore only to be regarded as descriptive, in no way as in any way limiting in any way.

The complete disclosure of all of the applications, patents and publications mentioned above and below, in particular the corresponding application DE 101 32 280.1, filed on July 4, 2001, is incorporated by reference into this application.

Examples

The following implementation example relates to the use of ampicillin-sensitive strains with a plasmid-localized mutated ampicillin resistance gene (raster thrust strain and base exchange strain) and the feM promoter-controlled reporter system β-galactosidase. The plasmid is introduced into the test strain E. coli EE122 or Salmonella typhimurium TA1535. The chemiluminometric measurement is described as the detection method. The strain for the detection of raster shear mutations is referred to in the following and in the result examples given as 4S or 6S, the strain for the detection of base exchange mutations as 3S or 14S.

The test strains used in the implementation example carry a plasmid with a pBR322 [Sutcliffe, JG; Cold Spring Harbor Symp. Quant. Biol. 43, 1997, 77-90] derived replication origin (pMB1). Additional cloning of the par ('partition') region from pSC101 [Meacock, PA and Cohen, SN, Cell 20, 1980, 529-542] ensured the stable inheritance of the plasmid on the daughter cells. As The plasmid pBR328 (National Institute of Genetics, Yata, Japan) served as the donor plasmid for the second resistance gene (in the example given, the cam resistance gene). The muc> 4β operon was taken from plasmid pGW1700 [Perry, KL and Walker, GG, Nature 300, 1982, 278-281]. For the stringently controlled expression of the ß-

Galactosidase reporter gene, the lacZY gene sequence had to be cloned in the correct reading frame behind the promoter / operator region (tet ^{p / 0} ) of the teM gene into the plasmid pASK75 [Skerra, Gene 151, 131-135, 1994]. This additionally contains the gene coding for the fef repressor (TetR) transcriptionally fused to the constitutively expressed bla gene (ampicillin resistance), whereby a strong repression of the tetA promoter is achieved [Skerra, Gene 151, 131-135, 1994 ]. The plasmid pMC1403, in which the promoter region and the first eight amino acids (including the start ATG) of the β-galactosidase are missing, served as the source for the lacZY genes [Casadaban et al., J. Bacteriol. 143, 1980, 971-980]. Since only a few restriction sites are available for the isolation of the sequence from pMC1403, the / acZY genes were first cloned into the pGFPuv vector. The restriction enzymes of the 'Multiple Cloning Site' (MCS), which are now located at the 5 'end of the lacZ sequence, then enabled directed cloning in the reading frame behind the start ATG of the teM gene.

By means of targeted mutagenesis, the base substitution or raster shear mutation was introduced into the ß-lactamase gene. As an example of base substitution, a transition from adenine to guanine was carried out in the active center 67 amino acids after the start ATG. As a result, the amino acid glycine was introduced instead of the amino acid serine (5'-AGC-3 '- 5'-GGC-3') and, as a result, the β-lactamase was reversibly inactivated. As another example of base substitution

(Strain 14S) became 66 amino acids after the start ATG one

Transversion from thymine to guanine made. As a result, the amino acid arginine was introduced instead of the amino acid methionine (5'- ATG-3 '→ 5'-AGG-3'). As an example of a raster shear mutation (Samm 4S), the bases guanine and cytosine were introduced in an alternating GC box nine amino acids after the active serine residue, resulting in 44 amino acids after the mutation resulting in a stop codon (TGA). An additional guanine was integrated in a further raster shear mutation strain (strain 6S) 17 amino acids after the active center, resulting in a stop codon 19 amino acids after the mutation. In all cases the ß-lactamase was reversibly inactivated. The plasmid was isolated into the Escherichia coli EE122 strain [Δ (lac proB) Δ (uvrB gal bio) ara thi rfa] [Eisenstadt, E., Warren, AJ, Porter, J., Atkins, D. and Miller, JH, Proc , Natl. Acad. Be. USA 79, 1982, 1945-1949].

1. Pre-culture

The bacteria (3S, 14S, 4S or 6S) are overnight (or at least for 3 to 4 hours) in an incubator with shaking at 37 ° C in Luria-Bertani (LB) medium with the addition of the antibiotic to maintain the plasmid incubated. The next morning (or after 3 to 4 hours) the bacteria are adjusted to a cell count of approx. 2.5 x 10 ⁷ (corresponds to E ₆₀ o of approx. 0.05) with LB medium and until incubation with the Test substances kept on ice.

2. Preparation of the test plates and incubation 2.1 Pre-incubation phase

The pre-incubation can e.g. with 24Well or 96Well test plates.

In the case of incubation without an S9 mix, 10 μl of different concentrations of the test substance, the negative control substance (solvent control) and the positive control substance are added to 1 ml of the bacteria set in accordance with paragraph 1. For substances that need S9 to express their mutagenic potential, an S9- Kofaktormix and S9 added. The cofactor mix is always made fresh on the test day according to Maron and Arnes (Maron and Arnes; Mutation Res. 113, 1983, 173-215). For incubation with S9, 1 ml of the bacteria set according to paragraph 1 is presented. 70 μl cofactor mix and 10 μl S9 are added. Then add 10 μl of the test sample. The approach is mixed well. This is followed by a 90-minute preincubation at 37 ° C with shaking.

2.2 Selection phase

After the pre-incubation, the test batches are diluted 1:10 with LB medium (+ 50 μg / ml ampicillin) and for 6 (short-term test at 37 ° C) to 16 hours (long-term test at 28 - 30 ° C) in 24Well, 96Well or 384Well Incubated plates.

3. Induction of the reporter system

For the induction of the reporter system, the batches with 100-200 ng / ml

Anhydrotetracycline was added and incubated at 37 ° C for 1-2 hours.

4. Measurement of mutagenicity

For the luminometric enzyme test of the reporter system, 60 μl of the bacterial suspension are mixed with 90 μl lysis buffer and 30 μl substrate solution (Galacton-Plus ^® ; Applied Biosystems, PE Deutschland GmbH). To stop the reaction after 30 min incubation at 28 ° C, 120 ul stop solution are added. Then 100 ul of

Overall approach taken for the luminometric measurement. After automatic injection of 100 μl luminescence enhancer solution (Luminescent Enhancer, ^Emerald® ; Applied Biosystems, PE Deutschland GmbH), each batch is measured in the luminometer after a waiting time of 2 seconds over a period of 5 seconds. To measure the chemiluminescence, 96-well luminometers (e.g. Labsystems, Luminoscan) or single-channel luminometers (e.g. Berthold, Lumat) can be used.

5. Toxicity control

The approach to toxicity control is carried out analogously to the approach to mutagenicity testing. A non-reversible strain that is identical in terms of the other genotype can be used as the test strain. Toxicity control can also be performed on the revertible test strains. The approach to this is analogous to the approach to mutagenicity testing. After 90 min of pre-incubation without selection agent, the optical density of the test batches (E _Θ OO nm) is measured immediately. If the measured E600 values of the substance batches are lower than those of the control batches, then there is cytotoxicity.

Indications of the toxicity of a mutagenic substance can also be drawn from the curve shape of the rlu values. One can speak of an increasingly toxic effect of a test substance as soon as the curve of the rlu values begins to flatten and possibly decreases with increasing substance concentrations.

6. Evaluation

The raw data (rlu values: relative luminescence units) are transferred to an MS Excel evaluation program. The mean values of the rlu minus the blank value control and the ratios of the test approaches versus control approaches are calculated.

In the example presented here, a substance is considered to be mutagenic if the signal / background ratio reaches a factor of 2 at at least 2 concentrations and there is a concentration-effect relationship.

Result example 1

Short-term test: pre-incubation: 90 min .; Selection phase: 6 hours

Long-term test: pre-incubation: 90 min .; Selection phase: 16 hours

Result example 2

Long-term test and color change detection (bromocresol red)

If the long-term test is carried out, the revertants can also be detected via a color change (by adding, for example, bromocresol red to the selection medium). After the pre-incubation, the bacteria are diluted with LB medium (+ ampicillin + bromocresol red) and incubated for 16-18 hours in 24-well, 96-well or 384-well plates at 37 ° C. There is no induction of the reporter gene. The evaluation is carried out either by counting the "positive" (in the case of bromocresol red the yellow) wells or by measurement with a photometer.

* Decrease in positive wells due to cytotoxicity at high NQO concentrations

Result example 3:

Long-term test and color change detection (ß-lactamase activity)

If the long-term test is carried out, the detection of the revertants can also be carried out via direct detection of the β-lactamase activity recovered after back mutation. After the pre-incubation, the bacteria are diluted with LB medium (+ ampicillin) and incubated for 16-18 hours in 24-well, 96-well or 384-well plates at 37 ° C. There is no induction of the reporter gene. After adding 50μl ß-lactamase substrate (e.g. nitrocefin [test concentration 50 μg / ml]) the test batches are 5 to 10min. incubated.

The evaluation is carried out either by counting the 'positive' wells or by measurement with a photometer (nitrocefin: E492 nm).

Claims

Expectations

1. Isolated prokaryotic test strain for the detection of mutagens at least comprising a) a revertible mutated selection marker in the form of a

Resistance gene against bacteriotoxic or bacteriostatic substances or influences; b) a reporter system, the expression of which can be detected selectively in reverted bacteria.

2. Test strain according to claim 1, characterized in that the selection marker also serves as a reporter system.

3. Test strain according to one of claims 1 or 2, characterized in that the selection marker is an antibiotic resistance gene against a bacteriolytic antibiotic or inhibiting protein biosynthesis.

4. Test strain according to one or more of claims 1 to 3, characterized in that the selection marker is a

Tetracycline resistance gene, an ampicillin resistance gene, a kanamycin resistance gene, a chloramphenicol resistance gene or a streptomycin resistance gene.

5. Test strain according to one or more of claims 1 to 4, characterized in that the reporter system in its expression leads directly or indirectly to a color reaction, a luminescence or fluorescence signal.

6. Test strain according to one or more of claims 1 to 5, characterized in that the β-galactosidase, β-lactamase or luciferase gene or operator is used as the reporter system.

7. Test strain according to one or more of claims 1 to 6 additionally having one or more of the following properties: a) a cell wall permeable to large, lipophilic molecules; b) a defective excision repair c) a functional regulation unit of the SOS system d) the mutator genes umuDC and / or mucAB

8. A method for the detection of mutagens essentially characterized by the following method steps: a) providing at least one test strain according to one of claims 1 to 7; b) incubation of the test strain with the potential mutagen; c) selection of revertants; d) optionally induction of the reporter system; e) detection of the gene product of the reporter system and / or detection of the growth of the reverted strains.

9. The method according to claim 8, characterized in that in step e) the detection of the gene product of the selection marker as detection of the

Gen product of the reporter system.

10. The method according to claim 8 or 9, characterized in that the detection of the growth in step e) is carried out by means of a pH indicator in the medium.

11. Test kit for carrying out mutation tests essentially containing one or more test strains according to one of claims 1 to 7.

12. Use of at least one test strain according to one of claims 1 to 7 for HTP screening.