JP3277436B2 - Mutagen detection method using luminescent gene - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a luminescent short-term mutagen detection method.

[0002]

2. Description of the Related Art At present, many mutation test methods using microorganisms have been reported as short-term screening methods for carcinogenic substances. Among them, the reverse mutation test method, Am
The es test is the most widely used. However, this test method has a problem that it takes about 3 days to measure and requires aseptic operation. Recently, a short-term mutagen detection method (umu test, SOS chromo test) for detecting DNA damage, unlike the Ames method for detecting mutagenicity, was reported (Oda, Nakamura et al., "Evalution of the
new system (umu-test) for the detection of environ
mental mutagensand carcinogens ", Mutation Researc
h, 147 (1985) 219-229, Japanese Patent Publication No. 4-12118).

[0003] In these methods, the SOS response induced at the time of DNA damage is determined by using one of the SOS genes, umuD,
This is a method for detecting DNA damage and mutagenicity by measuring the expression level of β-galactosidase by ligating the lacZ gene downstream of the C gene or sfiA gene. More specifically, after culturing Salmonella (Umu test) and Escherichia coli (SOS Chromotest) into which the above-described genes have been introduced in the presence of a test substance, toluene or chloroform and a sulfonate such as sodium dodecylbenzenesulfonate are used. An aqueous solution of a surfactant is added to perform cell wall destruction treatment.

Then, for the purpose of measuring the β-galactosidase activity, an aqueous solution of 2-nitrophenyl-β-D-galactopyranoside is added, the enzyme reaction is carried out at 28 ° C., and after several tens minutes, the reaction is stopped with sodium carbonate. I do. The optical density of the enzyme reaction solution was measured at two wavelengths (420, 550 nm) and the mirror method (Miller. JH, Experiments in molecular genetic
s, Cold Spring Harbor Laboratory, 1972) to calculate β-galactosidase activity.

The above-mentioned umu test and SOS chromo test can be detected much more quickly than the Ames method, but they are colorimetric detections, so their detection sensitivity is low, and the detection time still requires about 7 to 8 hours. In particular, these tests
The detection sensitivity for nitroarenes and polycyclic aromatic compounds is low, which is a major problem.

[0006]

SUMMARY OF THE INVENTION Accordingly, the present invention provides a SOS gene DN having a high detection sensitivity and a short detection time.
It is an object of the present invention to provide a method for detecting a mutagenic substance using sensitivity to A damage.

[0007]

Means for Solving the Problems The present inventors have conducted various studies to solve the above-mentioned problems, and as a result, have found that a group in which a gene that expresses luciferase activity is arranged downstream of an SOS gene that is sensitive to DNA damage. When the microorganism transformed with the transgene is cultured in a medium containing a mutagenic substance, a gene having luciferase activity is also expressed at the same time as the expression of the SOS gene, and the light generated thereby is measured. The present inventors have found that a mutagenic substance can be detected or measured with high sensitivity in a short time, and completed the present invention.

Accordingly, the present invention provides a recombinant gene comprising an SOS gene expressed upon DNA damage and a gene having luciferase activity arranged downstream of the SOS gene. The present invention also provides a host microorganism transformed with the gene. The present invention further comprises culturing the host microorganism in a medium containing a test sample, and then measuring the luminescence due to the expression of the gene expressing the luciferase activity, wherein the mutagenicity in the test sample is measured. A method is provided for determining the presence or absence of a substance or measuring the amount of a substance.

In the present invention, in order to express a gene that expresses luciferase activity depending on DNA damage, the SOS gene is used as an expression control means. Therefore, the SOS gene only needs to be expressed at the time of DNA damage, and only needs to contain a control portion called an SOS box. Therefore, in the present invention, the “SOS gene expressed upon DNA damage” means a gene containing a control site called the SOS box, and may be the SOS box itself, or any DNA fragment containing the same. It may be.

[0010] SOS genes include umu genes, such as the umuC and umuD genes, and the sfi gene.
A gene and the like are included, but not limited thereto. Of these genes, the umuC and umuD genes are Pr
o. Natl. Acad. Sci. USA, Vol. 82, 4336-4340 (1985)
And can be easily obtained based on these descriptions. Also, a plasmid pSK1002 containing a fusion gene of the umuD gene and the lacZ gene.
Is described in H. Shinagawa et al., Gene, 23, 167 (1983), and the umuD gene can be easily obtained from this plasmid.

[0011] As a gene expressing luciferase activity,
Can be of various origins.
For example, North American fireflies (Photinus pyrali
s) -Derived luciferase gene (luc gene)
The vector entered is Picker Gene ^(TM)Cassette vector and
Sold by Toyo Ink Manufacturing Co., Ltd.
Convenient to use.

The host microorganism may be any microorganism that can express the SOS gene when its DNA is damaged by a mutagen, that is, a microorganism having an SOS function. For example, Escherichia coli ( Escheric)
hia coli), for example the CSH26 strain, Salmonella (Salmonella typhimuriu
m ), for example, the TA1535 strain, 1538 strain, etc., and Bacillus , for example, B. subtilis, and the like.

A recombinant gene comprising the SOS gene and a gene expressing luciferase activity is linked to a gene expressing luciferase activity downstream of the SOS box of a DNA fragment containing at least the SOS box of the SOS gene. By doing so, it can be manufactured. This ligation can be performed using DNA ligase according to a conventional method. In order to introduce this recombinant gene into a host microorganism, the recombinant gene must be present in a vector.
BR-based plasmids, pUC-based plasmids and the like can be used.

Transformation of a host microorganism with an expression vector may be performed by a conventional method used for transforming a microorganism, for example, a bacterium. According to the present invention, to detect or measure a mutagenic substance in a test sample, the test sample is mixed with a medium, and the transformed host microorganism is cultured in the medium. The culture is usually performed for 1 to 3 hours. The cultured cells are then collected and the cell wall is disrupted to release the expression product, luciferase, from the cells. This cell destruction can be easily carried out according to a conventional method, for example, using a cell wall lysing agent, for example, Triton X, SDS, which is a surfactant, or by mechanical treatment such as ultrasonic treatment.

Next, the cell lysate is centrifuged, for example, to obtain a luciferase-containing supernatant.
This is subjected to light emission processing. This luminescence treatment is performed by adding necessary components such as a substrate (for example, luciferin) and a coenzyme. The light thus generated can be detected according to a conventional method. According to the method of the present invention, all mutagenic substances can be detected. The mutagenic substance is finally introduced into the medium as a solution, but it can be measured even if it is a gas or a solid that can be dissolved in the medium.

[0016]

According to the conventional Ames test, it takes a predetermined time (at least 30 minutes or more) to obtain sufficient color development for measurement, whereas in the present invention, sufficient light emission for measurement is instantaneously emitted. In terms of obtaining and the like, the method of the present invention greatly reduces the detection time. Also, the method of the present invention,
Since luminescence detection is used, the volume of the sample required for luminescence measurement can be reduced, so that a mutagenic substance can be detected with a sensitivity about 100 times higher in terms of absolute amount than conventional colorimetric detection. Furthermore, since the detection is based on luminescence, the dynamic range of the measurement is widened, and the minimum detection concentration of the mutagenic substance can be detected at least four times or more at a low concentration side with high sensitivity. In particular, nitroarenes and polycyclic aromatic compounds can be detected with high sensitivity at a minimum concentration of about 10 times lower than the minimum detection concentration.

[0017]

Next, the present invention will be described more specifically with reference to examples. Embodiment 1 FIG. Measurement in Escherichia coli
A construction method is employed in which a umuC-luc fusion protein is produced by a fusion gene of a uC gene and a luciferase gene (hereinafter abbreviated as luc), but a gene encoding a polypeptide having luciferase activity is a member of the SOS gene group. As long as it is under control, the construction method does not necessarily need to be such that a fusion protein is produced.

As the SOS gene, umuD, C-la
Plasmid pSK1002 with the cZ fusion gene "H.
Shinagawa, T.Kato, T.Ise, K.Makino, A.Nakata,Gen
e,twenty three, 167 (1983) ". Rushi
A polypeptide having ferase activity as an encoding gene;
Picka Gene manufactured by Toyo Ink Manufacturing Co., Ltd.^TMCassette
North American fireflies (Photoninus py)
lucis) (lucis).

As a bacterium to be introduced, Escherichia coli (CSH26 "Mi
ller, J.H., Experiments in Molecular Genetics, Col
d Spring Harbor Laboratory, 1972 ": F^-ara del (la
c-pro) thi), Salmonella (TAl535 `` Ames, B.N., J. Mc
Cann, E. Yamasaki,MutationRes.,31, 347, (1975)
 : HisG46, Δgal, Δchl, Δbio, ΔuvrB, rf
a ^-, SJ10002: r^-, m⁺) Was used.

As a luminescent substrate and a cell lysing agent used in the luminescence measurement, a luminescence kit (Picagene ^™ ) manufactured by Toyo Ink Co., Ltd. was used. The composition of the luminescent substrate was 20 mM Tricine,
1.07mM (MgCO ₃ ) ₄ Mg (OH) ₂・ 5H ₂ O, 2.67mM MgSO ₄ , 0.1mM
EDTA, 33.3 mM DTT, 270 μM CoenzymeA, 470 μM luc
iferin, 530 μM ATP. The composition of the cell lysing agent is 25 mM Tri
s-phosphate (pH 7.8), 2mM DTT, 2mM 1,2-diaminocyclo
hexane-N, N, N, N-tetraacetic acid, 10% glycerol,
1% Triton X-100. As the luminescence measuring device, a universal photon counting system manufactured by Hamamatsu Photonics Co., Ltd. was used.

E. coli CS containing pSK1002 vector
H26 containing LB medium containing ampicillin (Bacto tryptone 1%, Bacto yeast extract 0.5%, NaCl
1%), alkali-SDS method "Birnboim, H. et al.
C., Doly, J., Nucl . Acids Res ., 11 , 1513 (1979) ", prepared a large amount of the pSK1002 vector. The vector was cut with restriction enzymes HindIII and AvaI and um
A DNA fragment (A) of about 7.2 Kb containing the u gene was cut out. Fragment (A) was converted to Alkaline Phospha.
Dephosphorylation treatment was carried out using Tase (E. coli C75).

The vector containing the firefly-derived luc gene was transformed into Escherichia coli JM109 "Messing, J., Gene , 33 , 119 (198
5) was transformed. The transformed Escherichia coli was cultured in an LB medium containing ampicillin, and then subjected to alkaline-SDS method.
The vector containing uc was prepared in large quantities. Restriction enzyme Hin
The vector was cut with dIII and StuI to cut out a DNA fragment (B) of about 1.7 Kb containing the luc gene.

Fragment (A) and fragment (B) were combined with DNA Lig
Using Ligation Kit (Takara Shuzo), the ligation was followed by digestion with the restriction enzyme StuI. Then, DNA B
After performing blunt-end treatment using a lunting kit (Takara Shuzo), self-ligation was performed with a DNA Ligation Kit to construct an expression vector. In order to produce the umuC-luc fusion protein, the expression vector constructed above was cut with the restriction enzyme HindIII. After performing blunt-end treatment with DNA Blunting Kit, DNA Ligation Kit
Was used to construct a luminescent vector for producing an umuC-luc fusion protein.

CSH26 was cultured overnight in LB medium. L
1/100 volume of the above culture solution was added to the B medium,
The cells were cultured until the turbidity (OD ₆₀₀ ) at nm became about 0.4. 5 ml of the culture solution is centrifuged, and 5 ml of the precipitated fraction (cells) is centrifuged.
Was suspended in a 30 mM aqueous solution of CaCl ₂ and left on ice for 45 minutes. Centrifugation was performed again, and the cells were clarified in 0.4 ml of 30 ml.
Suspended in mM CaCl ₂ .

To 100 μl of the competent cells of CSH26 thus prepared, about 200
ng was added and left on ice for 30 minutes. After treatment at 42 ° C. for 2 minutes, the mixture was left at room temperature for 5 minutes. 1 ml of LB medium was added and incubated at 37 ° C. for 1 hour. This solution was seeded on an LB plate containing 50 μg / ml of ampicillin.

The CSH26 luminescent vector transformant was cultured overnight in an LB medium containing 20 μg / ml of ampicillin. This culture solution was inoculated into LB medium at 1/50 volume, and
℃ up to the logarithmic growth phase (OD ₆₀₀ = 0.2~0.3) were cultured. This bacterial solution was dispensed into test tubes in an amount of 29 μl each, and 2- (2-Furyl) -3- (5-n
intro-2-furyl) -acrylamide
(Hereinafter abbreviated as AF-2) (1 μl), and the mixture was incubated at 37 ° C. for 2 hours.

This bacterial solution was centrifuged, and 20 μl of a cell lysing agent was added to the precipitated fraction (cells) and suspended. The suspension was treated 5 times for 30 seconds using an ultrasonic crusher (Tosho Denki) and centrifuged. 100 μl of a luminescent substrate was added to 10 μl of the supernatant fraction, and luminescence was measured. The measurement was performed for 8.192 seconds (2 milliseconds × 40
The number of photons during 96) was counted, and the luciferase activity was determined. The results are shown in FIG.

[0028]Embodiment 2. FIG. Salmonella (TA153
Measurement in 5) Competent of SJ10002 in the same manner as in Example 1.
A cell was prepared. Each of TA1535 in LB medium
Cultured overnight. 1/100 volume of the above culture solution in LB medium
And the turbidity at 600 nm (OD₆₀₀) To about 0.4
Cultured until 5 ml of the culture solution is centrifuged and the precipitate fraction
(Bacteria) in 5 ml of 10 mM CaCl_Two , 10 mM MnC
l_Two , 10 mM MgCl_Two Suspend in aqueous solution and ice for 45 minutes
Left inside. Perform centrifugation again, and
10 mM CaCl_Two , 10 mM MnCl_Two , 10mM
gCl_Two Suspend in aqueous solution and compete with TA1535
A cell was prepared.

Competent cell 100 of SJ10002
About 200 ng of the luminescent vector (prepared in Example 1) was added to μl, and left on ice for 30 minutes. After treatment at 42 ° C. for 2 minutes, the mixture was left at room temperature for 5 minutes. 1 ml of LB medium was added and incubated at 37 ° C. for 1 hour. This solution was seeded on an LB plate containing 50 μg / ml of ampicillin. The luminescent vector introduced into SJ10002 was prepared by the alkaline-SDS method, and TA1 was prepared in the same manner as described above.
Transformation into 535 was performed.

Using a luminescent vector transformant of TA1535, the same method as in Example 1 (TG instead of LB medium) was used.
A medium (using 1% tryptone, 0.5% salt, 0.2% glucose, 20 μg / ml ampicillin) in A medium
The luminescence measurement by F-2 was performed. The results are shown in FIG.
The comparative examples shown below were performed to compare the sensitivity of the present invention with that of the conventional method.

Comparative Example 1 Escherichia coli (CSH26 / pSK1002) into which plasmid pSK1002 was introduced was used. 0.1 mol / l potassium phosphate buffer (pH 7.0)
Then, 0.75 g of potassium chloride, 0.246 g of magnesium sulfate and 2 ml of 0.05 mol / l 2-mercaptoethanol were added to and dissolved in 11 to prepare a Z-Buffer.

CSH26 / pSK1002 was cultured overnight in an LB medium containing 20 μg / ml of ampicillin. This culture was inoculated into the same medium at 1/50 volume and cultured at 37 ° C. until the logarithmic growth phase (OD ₆₀₀ = 0.2-0.3). 2.9 ml of this bacterial solution was dispensed into test tubes, and 0.1 ml of a predetermined concentration of AF-2 was added to each, followed by incubation at 37 ° C. for 2 hours. To 0.2 ml of this bacterial solution, add Z-Buffer
l. 8 ml, 50% of 0.1% SDS, chloroform 10
μl was added and stirred for 5 seconds. Next, 0.4 ml of a 4 mg / ml aqueous solution of 4-nitrophenyl-β-D-galactopyranoside (ONPG) dissolved in 0.1 M phosphate buffer (pH 7.0) was added, and the mixture was reacted at 28 ° C. for 10 minutes. 1mol
The reaction was stopped by adding 1 ml of a 1 / l sodium carbonate solution. Then, the absorbance at 420 nm was immediately measured. The results are shown in FIG.

Comparative Example 2 Salmonella harboring plasmid pSK1002 (TA1535 / pSK100
2) was used. Using TA1535 / pSK1002,
A method similar to that of Comparative Example 1 (TGA medium (1% tryptone, 0.5% salt, 0.2% glucose)
%, 20 μg / ml ampicillin).
Was used to measure the induced β-galactosidase activity. The results are shown in FIG.

As shown in FIGS. 1 and 2, in a strain in which a recombinant plasmid having a luminescent gene was introduced into CSH26, good doF was obtained from about 1 ng to about 100 ng of AF-2.
A se-response curve was given. In a strain in which a recombinant plasmid having a luminescent gene was introduced into TA1535, AF-2 exhibited a good d from about 0.1 ng to about 1 ng.
An o-response curve was given. Compared with the results of the conventional method (Comparative Example), the method of the present invention shows that CSH
Both strains 26 and TA1535 were about 100 times better in sensitivity.

Example 3 Salmonella (TA1535)
Obtained from the liver of measuring metabolic activation enzyme rats with phenobarbital and 5,6
Benzoflavone-treated S9 fraction (manufactured by Oriental Yeast Co., Ltd.)
An S9 mix mixed with ctor-I (manufactured by Oriental Yeast Co., Ltd.) was used.

Test sample AF-2,4-nitroquinoline 1-oxide (hereinafter referred to as 4)
NQO), 1-nitropyrene (hereinafter abbreviated as 1-NP), 2-aminoanthracene (hereinafter abbreviated as 2-AA), and benzo [a] pyrene (hereinafter abbreviated as BaP) were used. The TA1535 luminescent vector transformant obtained in Example 2 was cultured overnight at 37 ° C. in a TGA medium. This culture solution was inoculated in a 1/50 volume in a TGA medium and cultured at 37 ° C. for 1.5 hours. This culture was diluted with TGA medium so that OD ₆₀₀ = 0.1. 1.45 ml of this diluted culture solution
Aliquots into test tubes, each having a predetermined concentration of test sample 5
0 µl was added and the cells were cultured at 37 ° C for 2 hours. When a metabolic activator was required, 0.25 ml of the S9 mix and 50 μl of the test sample were added to 1.2 ml of the diluted culture, and the mixture was cultured at 37 ° C. for 2 hours.

After the culture, 0.5 ml of the bacterial solution was centrifuged, and 50 μl of a cell lysing agent was added to the precipitated fraction (cells) and suspended. In a 96-well plate for microluminoreader, 10 μl of the suspension was added.
100 μl of a luminescent substrate was added, and the amount of luminescence was measured with a luminescence measuring device (Microlumino Reader (MLR-100) manufactured by Corona). Further, the OD ₆₀₀ of the culture solution was measured. The activity (Intensity / OD ₆₀₀ ) was determined by measuring the amount of luminescence for 10 seconds and dividing the value by the value of OD ₆₀₀ . The measurement results are shown in FIGS.

Comparative Example 3 TA1535 / pSK1002 was added to a TGA medium for 3 nights.
The cells were cultured at 7 ° C. This culture solution was inoculated in a 1/50 volume in a TGA medium and cultured at 37 ° C. for 1.5 hours. This culture was diluted with TGA medium so that OD ₆₀₀ = 0.1.
1.45 ml of the diluted culture solution was dispensed into test tubes, and 50 μl of a test sample having a predetermined concentration was added to each, and the mixture was cultured at 37 ° C. for 2 hours. When a metabolic activating enzyme is required, 0.25 ml of S9 mix and 1.2 ml of test sample
0 μl was added, and the cells were cultured at 37 ° C. for 2 hours. O of this bacterial solution
_D600 was measured.

In the remaining 0.1 ml of the bacterial solution, add Z-Buffer
0.9 ml, 50% of 0.1% SDS, chloroform 1
0 μl was added and stirred for 5 seconds. Next, 0.2 ml of a 4 mg / ml aqueous solution of ONPG dissolved in 0.1 M phosphate buffer (pH 7.0) was added, and reacted at 28 ° C. for 10 minutes.
The reaction was terminated by adding 0.5 ml of an ol / 1 sodium carbonate solution. Then, the absorbance at 420 nm and 550 nm was measured immediately. The activity (unit) was determined by the following equation based on the Miller method.

[0040]

(Equation 1) As shown in FIGS. 3 and 5, when the luminescence measurement method of the present invention was used, the dynamic range of the measurement of each test sample was several times wider than that of the conventional colorimetric detection.

As shown in FIGS. 4 and 5 and Table 1, when the luminescence measurement method of the present invention was used, the minimum detection concentration of each test sample was at least about four times lower than the conventional colorimetric detection. . In particular, for nitroarenes (1-NP) and polycyclic aromatic compounds (BaP), the minimum detection concentration is about 1
The concentration became 0 times lower. The examples described below were carried out in order to clarify the mechanism of high-sensitivity detection, such as expansion of the dynamic range of the measurement of the present invention and reduction of the minimum detection concentration.

Example 4 A predetermined amount of luciferase dissolved in 10 μl of a cell lysing agent and 100 μl of a luminescent substrate were added to a 96-well plate for a microlumino reader, and the amount of luminescence for 10 seconds was determined. The enzyme amount (luciferase amount) is plotted on the horizontal axis, and the corresponding activity (light emission amount) is plotted on the vertical axis, and the results are shown in FIG.

To a predetermined amount of β-galactosidase dissolved in 0.1 ml of water, 0.9 ml of Z-Buffer, 0.1% S
50 μl of DS and 10 μl of chloroform were added, and 5
Stirred for seconds. Next, a 0.1 M phosphate buffer (pH 7.
0.2 ml of a 4 mg / ml aqueous solution of ONPG dissolved in 0) was added and reacted at 28 ° C. for 10 minutes, and 0.5 ml of a 1 mol / 1 sodium carbonate solution was added to stop the reaction. And
Immediately, the absorbance at 420 nm was measured. β-galactosidase activity (unit) was determined by the following equation.

Activity = 1000 × OD _{420 The} enzyme amount (β-galactosidase amount) is plotted on the horizontal axis, and the corresponding activity (β-galactosidase activity) is plotted on the vertical axis, and is shown in FIG. Further, Example 3, Comparative Example 3,
The amounts of luciferase and β-galactosidase induced when each concentration of AF-2 was added were determined from FIG. 6 and are shown in FIG. From the results of FIGS. 6 and 7, the reason why high-sensitivity detection in the present invention became possible was inferred as follows.

The expression efficiency of luciferase is generally lower than that of β-galactosidase, and when no test sample is added, the latter expresses only about 1/10 ⁶ of the former. That is, a large amount of β-galactosidase is synthesized even when no test sample is added. This is thought to be due to the influence, but the response of the induction synthesis of β-galactosidase by the addition of the test sample is slow, and the relative ratio of the amount of the induced synthesis to the amount of the synthesis not induced is small. .

On the other hand, the response of the luciferase-induced synthesis by the addition of the test sample is fast, and the relative ratio of the amount of the induced synthesis to the synthesis amount independent of the induction is large. The speed of response of induction synthesis by the addition of a test sample of the luciferase gene means that the minimum detection concentration is low, and the magnitude of the relative ratio of the amount of induction synthesis to the amount of synthesis not dependent on induction is high. Is
This means that the dynamic range of the measurement is increased.

As described above, these phenomena and the effects of the present invention are brought about by the difference in expression efficiency between the β-galactosidase gene and the luciferase gene in the host Salmonella TA1535. It has been found. In addition, low expression efficiency means that it is difficult to detect with conventional colorimetric detection, indicating that the features of using high-sensitivity luminescence detection with luciferase produced the effects of the present invention. I have.

[0048]

[Table 1]

[Brief description of the drawings]

FIG. 1 shows the results of luminescence measurement and the conventional method (colorimetric method) obtained in Example 1 and Comparative Example 1.

FIG. 2 is a graph showing the results of luminescence measurement and a conventional method (colorimetric method) obtained in Example 2 and Comparative Example 2.

FIG. 3 shows the value obtained by dividing the response at each concentration of each test sample obtained in Example 3 and Comparative Example 3 by the response under the condition not including the test sample (Ratio) → Ratio
It is the figure which plotted (0-20) with respect to a test sample concentration.

FIG. 4 is a value obtained by dividing the response at each concentration of each test sample obtained in Example 3 and Comparative Example 3 by the response under conditions not including the test sample (Ratio) → Ratio
It is the figure which plotted (0-10) with respect to a test sample concentration.

FIG. 5 shows a value obtained by dividing the response at each concentration of each test sample obtained in Example 3 and Comparative Example 3 by the response under the condition not including the test sample (Ratio) → Ratio
It is the figure which plotted (0-10) with respect to a test sample concentration.

FIG. 6 shows luciferase obtained in Example 4 and β-
FIG. 4 is a diagram of a calibration curve showing the relationship between the amount of galactosidase enzyme and activity.

FIG. 7: Using the calibration curves obtained in Example 4, the amounts of luciferase and β-galactosidase induced when each concentration of AF-2 was added in Example 3 and Comparative Example 3 were calculated. It is the figure plotted with respect to density.

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁷ Identification code FI C12R 1:42) C12N 15/00 A (56) References JP-B-4-12118 (JP, B2) Edited by The Biochemical Society of Japan “New Chemistry Laboratory Lecture 14 Development, Differentiation, and Aging” Tokyo Chemical Industry Co., Ltd., p. 322-3 (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 15/00-15/70 BIOSIS (DIALOG)

Claims (2)

(57) [Claims] 1. A SOS gene expressed at the time of DNA damage, and a SOS gene transformed downstream from the recombinant gene comprising a gene expressing luciferase activity arranged downstream of the SOS gene.
Lumonella . 2. A test sample, wherein the Salmonella according to claim 1 is cultured in a medium containing the test sample, and then luminescence due to expression of the genes expressing the luciferase activity is measured. A method for determining the presence or absence of a mutagenic substance in a substance or measuring the abundance.