NUCLEIC ACID THAT INTERACTS WITH A RECEPTOR FOR ENDOCRINE DISRUPTING CHEMICALS AND USE THEREOF

Abstract

This invention provides a nucleic acid that enables evaluation of various endocrine disrupting actions of a very small amount of endocrine disrupting chemicals with high sensitivity. Such nucleic acid comprises a total of 20 to 60 nucleotides comprising the nucleotide sequence shown in SEQ ID NO: 1 and shows excellent responsiveness to various endocrine disrupting chemicals.

Claims

1. A nucleic acid comprising a total of 20 to 60 nucleotides comprising the nucleotide sequence shown in SEQ ID NO: 1.

2. The nucleic acid according to claim 1, wherein arbitrary 4 nucleotides (nnnn) comprised in the nucleotide sequence shown in SEQ ID NO: 1 are kmkk, provided that k indicates g or t and m indicates a or c.

3. The nucleic acid according to claim 1, wherein arbitrary 4 nucleotides (nnnn) comprised in the nucleotide sequence shown in SEQ ID NO: 1 are GCGG or TATT.

4. The nucleic acid according to claim 1, which comprises nucleotide sequences of given nucleotide lengths on the 3′ terminal side and the 5′ terminal side of the nucleotide sequence shown in SEQ ID NO: 1.

5. The nucleic acid according to claim 1, which consists of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 5, 8, 34, 37, 40, 43, 46, 49, and 52.

6. A vector comprising the nucleic acid according to claim 1.

7. The vector according to claim 6, wherein the nucleic acid is designated as a unit and a plurality of units of the nucleic acids are bound to each other.

8. The vector according to claim 6, which comprises a reporter gene on the 3′ terminal side of a sense strand of the nucleic acid.

9. A transformant comprising the nucleic acid according to claim 1 introduced into a host.

10. The transformant according to claim 9, wherein the nucleic acid is designated as a unit and a plurality of units of the nucleic acids are bound to each other.

11. The transformant according to claim 9, which comprises a reporter gene on the 3′ terminal side of a sense strand of the nucleic acid.

12. The transformant according to claim 9, which comprises a nucleic acid encoding a receptor for endocrine disrupting chemicals that interacts with the nucleic acid introduced thereinto.

13. The transformant according to claim 12, wherein the receptor for endocrine disrupting chemicals is a juvenile hormone receptor of arthropods.

14. The transformant according to claim 13, wherein the juvenile hormone receptor of arthropods is a juvenile hormone receptor of Crustacea or an insect.

15. The transformant according to claim 14, wherein the juvenile hormone receptor of Crustacea is a juvenile hormone receptor of daphnid.

16. The transformant according to claim 15, wherein the juvenile hormone receptor of daphnid is a protein (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 12; or (b) a protein consisting of an amino acid sequence having 70% or higher identity to the amino acid sequence shown in SEQ ID NO: 12 and having activity of a transcription factor for a juvenile hormone receptor.

17. The transformant according to claim 9, which further comprises a nucleic acid encoding a transcription-coupling factor introduced thereinto.

18. The transformant according to claim 9, wherein the host is a yeast.

19. A method for evaluating a test substance comprising: a step of bringing a test substance into contact with a transformant comprising the nucleic acid according to claim 1 and a reporter gene on the 3′ terminal side of a sense strand of the nucleic acid introduced into a host and expressing a receptor for endocrine disrupting chemicals that interacts with the nucleic acid; and a step of assaying the expression level of the reporter gene, wherein the interaction between the test substance and the receptor for endocrine disrupting chemicals is evaluated based on the expression level of the reporter gene.

20. The method of evaluation according to claim 19, wherein, when the expression level of the reporter gene is increased after the contact with the test substance, the test substance is determined as an agonist for the receptor for endocrine disrupting chemicals.

21. The method of evaluation according to claim 19, wherein the test substance is brought into contact with the transformant together with at least one substance selected from the group consisting of the endocrine disrupting chemicals interacting with the receptor for endocrine disrupting chemicals, hormones, and the agonist for the receptor for endocrine disrupting chemicals, and, when the expression level of the reporter gene is lower than the expression level measured when the substance is brought into contact by itself, the test substance is determined as an agonist for the receptor for endocrine disrupting chemicals.

22. The method of evaluation according to claim 19, wherein the nucleic acid is designated as a unit and a plurality of units of the nucleic acids are bound to each other.

23. The method of evaluation according to claim 19, wherein a nucleic acid encoding the receptor for endocrine disrupting chemicals is introduced into the transformant.

24. The method of evaluation according to claim 19, wherein the receptor for endocrine disrupting chemicals is a juvenile hormone receptor of arthropods.

25. The method of evaluation according to claim 24, wherein the juvenile hormone receptor of arthropods is a juvenile hormone receptor of Crustacea or an insect.

26. The method of evaluation according to claim 25, wherein the juvenile hormone receptor of Crustacea is a juvenile hormone receptor of daphnid.

27. The method of evaluation according to claim 26, wherein the juvenile hormone receptor of daphnid is a protein (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 12; or (b) a protein consisting of an amino acid sequence having 70% or higher identity to the amino acid sequence shown in SEQ ID NO: 12 and having activity of a transcription factor for a juvenile hormone receptor.

28. The method of evaluation according to claim 19, wherein the transformant further comprises a nucleic acid encoding a transcription-coupling factor introduced thereinto.

29. The method of evaluation according to claim 19, wherein the host is a yeast.

30. A kit for assaying endocrine disrupting chemicals comprising the vector according to claim 8.

Description

EXAMPLES

[0070] Hereafter, the present invention is described in greater detail with reference to the examples, although the technical scope of the present invention is not limited to the following examples.

[Preparation of Yeast Transformant]

[0071] In the examples, reporter plasmids, plasmids for transcription factors (receptors for endocrine disrupting chemicals), and plasmids for transcription-coupling factors were prepared as described below and introduced into budding yeast strains to prepare yeast transformants.

[Preparation of Reporter Plasmids]

[0072] Oligo DNA (SEQ ID NO: 3) comprising a response element consisting of the nucleotide sequence shown in SEQ ID NO: 2 (43 nucleotides) and oligo DNA (SEQ ID NO: 4) partially complementary to SEQ ID NO: 3 were prepared and annealed to each other. At the time of annealing, oligo DNAs were incubated at 95° C. for 2 minutes and temperature was then gradually lowered from 90° C. to 37° C. over the time of 30 minutes. When two sequences are “partially complementary” to each other herein, a region excluding several nucleotides on the 5′ terminal side of a nucleotide sequence identified by a sequence identification number is complementary to the same region of the other sequence.

[0073] After annealing, oligo DNA in which 1, 2, 3, 4, or 5 response elements had been consecutively annealed was purified. Subsequently, oligo DNA resulting from consecutive annealing of 1, 2, 3, 4, or 5 response elements shown in SEQ ID NO: 2 was inserted into the SpeI site of the pRW95-3 μlasmid having the β-galactosidase gene prepared in accordance with the method described in Wolf S S et al., Biotechniques, 20 (4), pp. 568-573, 1996.

[0074] Plasmids each comprising the response element (54 nucleotides) consisting of the nucleotide sequence shown in SEQ ID NO: 5, the response element (30 nucleotides) consisting of the nucleotide sequence shown in SEQ ID NO: 8, the response element (22 nucleotides) consisting of the nucleotide sequence shown in SEQ ID NO: 34, the response element (36 nucleotides) consisting of the nucleotide sequence shown in SEQ ID NO: 37, the response element (43 nucleotides) consisting of the nucleotide sequence shown in SEQ ID NO: 40, the response element (43 nucleotides) consisting of the nucleotide sequence shown in SEQ ID NO: 43, the response element (43 nucleotides) consisting of the nucleotide sequence shown in SEQ ID NO: 46, the response element (43 nucleotides) consisting of the nucleotide sequence shown in SEQ ID NO: 49, or the response element (43 nucleotides) consisting of the nucleotide sequence shown in SEQ ID NO: 52 inserted thereinto were prepared in the same manner. Plasmids were prepared in the same manner except that oligo DNA shown in SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 50, or SEQ ID NO: 53 was used instead of the oligo DNA shown in SEQ ID NO: 3, and oligo DNA partially complementary to SEQ ID NO: 6 (SEQ ID NO: 7), oligo DNA partially complementary to SEQ ID NO: 9 (SEQ ID NO: 10), oligo DNA partially complementary to SEQ ID NO: 35 (SEQ ID NO: 36), oligo DNA partially complementary to SEQ ID NO: 38 (SEQ ID NO: 39), oligo DNA partially complementary to SEQ ID NO: 41 (SEQ ID NO: 42), oligo DNA partially complementary to SEQ ID NO: 44 (SEQ ID NO: 45), oligo DNA partially complementary to SEQ ID NO: 47 (SEQ ID NO: 48), oligo DNA partially complementary to SEQ ID NO: 50 (SEQ ID NO: 51), or oligo DNA partially complementary to SEQ ID NO: 53 (SEQ ID NO: 54) was used instead of the oligo DNA shown in SEQ ID NO: 4. Thus, a plasmid comprising 3 consecutive repeats of the response element shown in SEQ ID NO: 5 inserted thereinto, a plasmid comprising 1, 2, 3, or 4 consecutive repeats of the response element shown in SEQ ID NO: 8 inserted thereinto, a plasmid comprising 1, 2, 3, 4, or 5 consecutive repeats of the response element shown in SEQ ID NO: 34 inserted thereinto, a plasmid comprising 1, 2, 3, or 4 consecutive repeats of the response element shown in SEQ ID NO: 37 inserted thereinto, a plasmid comprising 1, 2, 3, or 4 consecutive repeats of the response element shown in SEQ ID NO: 40 inserted thereinto, a plasmid comprising 1, 2, 3, or 4 consecutive repeats of the response element shown in SEQ ID NO: 43 inserted thereinto, a plasmid comprising 1, 2, or 3 consecutive repeats of the response element shown in SEQ ID NO: 46 inserted thereinto, a plasmid comprising 1, 2, 3, or 4 consecutive repeats of the response element shown in SEQ ID NO: 49 inserted thereinto, and a plasmid comprising 1, 2, or 3 consecutive repeats of the response element shown in SEQ ID NO: 52 inserted thereinto were prepared. The nucleotide sequences shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 34, and SEQ ID NO: 37 are located upstream of the Vrille gene of Daphnia magna, the nucleotide sequence shown in SEQ ID NO: 40 is derived from the nucleotide sequence shown in SEQ ID NO: 2 by substitution of 3 nucleotides outside the 5′ terminus of the C-box (CACGCG), the nucleotide sequence shown in SEQ ID NO: 43 is derived from the nucleotide sequence shown in SEQ ID NO: 2 by substitution of 3 nucleotides outside the 3′ terminus of the E-box (CACGTG), the nucleotide sequence shown in SEQ ID NO: 46 is derived from the nucleotide sequence shown in SEQ ID NO: 2 by substitution of 3 nucleotides outside the 5′ terminus of the C-box (CACGCG) and 3 nucleotides outside the 3′ terminus of the E-box (CACGTG), the nucleotide sequence shown in SEQ ID NO: 49 is derived from the nucleotide sequence shown in SEQ ID NO: 2 by substitution of 4 nucleotides in a region (a linker region) between the C-box (CACGCG) and the E-box (CACGTG), and the nucleotide sequence shown in SEQ ID NO: 52 is derived from the nucleotide sequence shown in SEQ ID NO: 2 by substitution of 3 nucleotides outside the 5′ terminus of the C-box (CACGCG), 3 nucleotides outside the 3′ terminus of the E-box (CACGTG), and 4 nucleotides in a region (a linker region) between the C-box (CACGCG) and the E-box (CACGTG).

[0075] For comparison with the reporter plasmid comprising the response element, a plasmid comprising the nucleic acid consisting of 16 nucleotides (i.e., CACGCGGCGGCACGTG) located in an upstream region of the Vrille gene of Daphnia magna inserted thereinto was prepared. Such 16 nucleotides include the E-box (CACGTG) and the C-box (CACGCG) serving as juvenile hormone response elements in various insects. In accordance with the method for preparing plasmids described above, a plasmid comprising 1, 2, 3, or 4 consecutive repeats of the nucleic acid consisting of 16 nucleotides inserted thereinto was prepared.

[0076] For further comparison, a plasmid comprising a region including the juvenile hormone response element of the Kr-hi gene of Drosophila melanogaster (DmJHRR) inserted thereinto was prepared. DmJHRR is described in He Q et al., J. Biol. Chem., 289 (40), pp. 27874-27885, 2014. A plasmid comprising 3 consecutive repeats of the DmJHRR sequence inserted thereinto was prepared in accordance with the method described above, except that oligo DNA comprising the DmJHRR sequence (SEQ ID NO: 15) was used instead of the oligo DNA shown in SEQ ID NO: 3 and oligo DNA partially complementary to SEQ ID NO: 15 (SEQ ID NO: 16) was used instead of the oligo DNA shown in SEQ ID NO: 4.

[0077] In order to compare reporter activity influenced by the presence or absence of the C-box (CACGCG) and the E-box (CACGTG) in the nucleotide sequence shown in SEQ ID NO: 2, a nucleotide sequence having a C-box different from the C-box in SEQ ID NO: 2 (SEQ ID NO: 25), a nucleotide sequence having an E-box different from the E-box in SEQ ID NO: 2 (SEQ ID NO: 28), and a nucleotide sequence having a C-box and an E-box different from the C-box and the E-box in SEQ ID NO: 2 (SEQ ID NO: 31) were prepared. Specifically, oligo DNA comprising the nucleotide sequence shown in SEQ ID NO: 25 (SEQ ID NO: 26) and DNA partially complementary to SEQ ID NO: 26 (SEQ ID NO: 27), oligo DNA comprising the nucleotide sequence shown in SEQ ID NO: 28 (SEQ ID NO: 29) and DNA partially complementary to SEQ ID NO: 29 (SEQ ID NO: 30), and oligo DNA comprising the nucleotide sequence shown in SEQ ID NO: 31 (SEQ ID NO: 32) and DNA partially complementary to SEQ ID NO: 32 (SEQ ID NO: 33) were prepared, and a plasmid comprising 4 consecutive repeats of the nucleotide sequence shown in SEQ ID NO: 25 inserted thereinto, a plasmid comprising 4 consecutive repeats of the nucleotide sequence shown in SEQ ID NO: 28 inserted thereinto, and a plasmid comprising 4 consecutive repeats of the nucleotide sequence shown in SEQ ID NO: 31 inserted thereinto were prepared.

[Preparation of a Plasmid Expressing a Transcription Factor]

[0078] In order to prepare a plasmid expressing a juvenile hormone receptor of Daphnia magna (hereafter, referred to as a “DampaMET plasmid”), a CEN6/ARS4 fragment was first prepared. A DNA fragment comprising the autonomously replicating sequence (ARS) and the centromere sequence (CEN) of yeast was amplified by PCR with the use of, as a template, the pYTβ reporter plasmid and the primers for CEN/ARS amplification: pUdp6 I-SceI CEN6 FW (SEQ ID NO: 17) and pUdp6 I-SceI ARS4 RE (SEQ ID NO: 18). PCR was carried out by repeating a cycle of 94° C. for 20 seconds, 58° C. for 20 seconds, and 72° C. for 1.5 minutes 35 times.

[0079] A PCR-amplified fragment and the pUdp6 μlasmid linearized by cleavage with the AatII restriction enzyme were introduced into wild-type yeast strains (Saccharomyces cerevisiae) W303a (MATa, ade2, his3, leu2, trpl, ura3) by the lithium acetate method. The pUdp6 μlasmid has a bidirectional promoter regions gal1 and gal10, a CYC terminator downstream of gal10, an ADH terminator downstream of gal1, and the uracil selection marker URA3 gene. A plasmid comprising the CEN6/ARS4 fragment inserted into pUdp6 via homologous recombination was extracted from yeast and introduced into the E. coli DH5α strain to prepare a low-copy episomal vector pUdp13. A recognition sequence of the 18-bp I-SceI restriction enzyme has been introduced into a site outside the CEN6/ARS4 sequence. After the target gene is cloned into the multicloning site, accordingly, the CEN6/ARS4 fragment is cleaved with I-SceI, and the resulting fragment can be readily converted into a genome insertion-type plasmid.

[0080] Subsequently, an open reading frame (ORF) of cDNA of the DampaMET gene was amplified by PCR using, as a template, cDNA of adult Daphnia magna carrying a resting egg and the primers for DampaMET: MET FW2 (SEQ ID NO: 19) and MET REV-3 (SEQ ID NO: 20). PCR was carried out by repeating a cycle of 94° C. for 20 seconds, 58° C. for 20 seconds, and 72° C. for 2.5 minutes 35 times.

[0081] cDNA of the amplified DampaMET gene and the pUdp13 μlasmid cleaved with the BamHI and HindIII restriction enzymes were introduced into the wild-type yeast strain W303a by the lithium acetate method. A plasmid prepared by this method, which comprises cDNA of the DampaMET gene inserted into a site downstream of the GAL10 promoter of the pUdp13 plasmid, was designated as pUdp13-DapmaMet. pUdp13-DapmaMet constructed in a yeast cell was recovered, introduced into the E. coli DH5a strain, and amplified.

[0082] Subsequently, pUdp13-DapmaMet was cleaved with the I-SceI restriction enzyme to obtain the CEN6/ARS4 sequence and converted into a genome insertion-type plasmid. Thus, the DampaMET plasmid comprising the DampaMET gene and a promoter upstream thereof was obtained.

[0083] A plasmid expressing a juvenile hormone receptor of Drosophila melanogaster (hereafter, referred to as a “DmMET plasmid”) was prepared in the same manner. At the outset, ORF of cDNA of the DmMET gene was amplified by PCR using, as a template, cDNA of Drosophila melanogaster larvae (Clontech) and the primers for DmMET: DmMET Fwd (SEQ ID NO: 21) and DmMET Rev (SEQ ID NO: 22). PCR was carried out by repeating a cycle of 98° C. for 10 seconds, 55° C. for 30 seconds, and 68° C. for 1.5 minutes 25 times.

[0084] cDNA of the amplified DmMET gene was cleaved with the SmaI and EcoRI restriction enzymes. cDNA of the cleaved DmMET gene was inserted into a region downstream of the GAL10 promoter of the pUdp6 μlasmid cleaved with the SmaI and EcoRI restriction enzymes. Thus, the DmMET plasmid comprising the DmMET gene and a promoter upstream thereof was obtained.

[Preparation of a Plasmid Expressing a Transcription-Coupling Factor]

[0085] In the example, in particular, a plasmid expressing a transcription factor comprising the Drosophila melanogaster Tai (DmTai) gene (hereafter, referred to as a “DmTai plasmid”) and a plasmid expressing a Daphnia magna transcription-coupling factor (SRC) (hereafter, referred to as a “DampaSRC plasmid”) were prepared and used. The DmTai plasmid was prepared in accordance with Ito-Harashima S et al., FEBS Open Bio., 7 (7): pp. 995-1008, 2017.

[0086] The DampaSRC plasmid was prepared in the manner described below. At the outset, ORF of cDNA of the Daphnia magna SRC (DampaSRC) gene was amplified by PCR using, as a template, cDNA of adult Daphnia magna carrying a resting egg and the primers for SRC: SRC-F1F-pESC (SEQ ID NO: 23) and SRC-F4short-pESC (SEQ ID NO: 24). PCR was carried out by repeating a cycle of 94° C. for 20 seconds, 58° C. for 10 seconds, and 72° C. for 7 minutes 35 times.

[0087] Subsequently, the pESC-Leu plasmid (Agilent technology) was cleaved with the SpeI and PacI restriction enzymes, and cDNA of the amplified DampaSRC gene was introduced into the wild-type yeast strain W303a by the lithium acetate method. Thus, the DampaSRC plasmid comprising cDNA of the DampaSRC gene inserted downstream of the gal1 promoter region of the pESC-Leu plasmid was obtained. The DampaSRC plasmid constructed in a yeast cell was recovered, introduced into the E. coli DH5a strain, and amplified.

[Preparation of Yeast Transformant]

[0088] At the outset, the cells of the budding yeast strain W303a were cultured in a YPD medium (1% yeast extract, 2% peptone, 2% D (+)-glucose) at 30° C. until the turbidity (O.D. 595) reached 0.7 to 0.8. The cultured cells were washed 2 times with sterile water and resuspended in a 0.1 mol/1 lithium acetate solution in an amount of 1/10 of the culture solution using a pipette. The cell suspension was fractionated in an amount of 100 μl each to 1.5-ml microtubes, and the supernatant was removed via centrifugation. A TE buffer (75 μl; 10 mM Tris, 1 mM EDTA, pH 8.0) containing 1 μg of the DampaMET plasmid linearized via treatment with the EcoRV restriction enzyme and 50 μg of carrier DNA (tradename: SALMON TESTESDNA for hybridization, SIGMA) was added, and 240 μl of a 50% polyethylene glycol 3350 solution and 36 μl of a 0.1 mol/1 lithium acetate solution were further added, followed by thorough mixing.

[0089] The mixture was incubated at 30° C. for 30 minutes, heat-treated at 42° C. for 22 minutes, and centrifuged at 9,000 rpm for 1 minute to harvest yeast cells from the mixture. The harvested yeast cells were suspended in 300 μl of sterile water, 100 ml of the suspension was applied to a selection medium prepared by adding 42 mg of tryptophan, 62 mg of leucine, and 2% agarose to the medium shown in Table 1 and Table 2, and culture was then performed. Uracil-nonrequiring yeast strains were selected as yeast transformants comprising the gal1 promoter and the cDNA region of the MET gene integrated into the chromosome.

[0090] Subsequently, the DampaSRC plasmid was introduced into the yeast transformant comprising the DampaMET plasmid introduced thereinto. As a selection medium for the yeast transformant, a culture medium prepared by adding 100 mg of tryptophan to the culture medium shown in Table 1 and Table 2 was used.

TABLE-US-00001 TABLE 1 Composition of pre-culture medium Components Amount Yeast nitrogen base w/o amino acids and ammonium sulfate 1.7 g (NH.sub.4).sub.2SO.sub.4 5 g Drop-out powder (Table 2) 1.3 g 5M NaOH 500 μl D(+)-glucose 20 g Water 11

[0091] Solid medium was prepared with the addition of 2% agar to the above composition, autoclaving, and transfer to a petri dish

TABLE-US-00002 TABLE 2 Composition of drop-out powder Components Amount Adenine 2.5 g L-Aspartic acid 6.0 g L-Histidine 1.2 g L-Arginine-HCl 1.2 g L-Methionine 1.2 g L-Lysine-HCl 1.8 g L-Glutamic acid 6.0 g L-Valine 9.0 g L-Serine 22.5 g L-Threonine 12.0 g

[0092] Subsequently, a reporter plasmid prepared by inserting the nucleotide sequence shown in SEQ ID NO: 2 into a yeast comprising the DampaMET plasmid and the DampaSRC plasmid introduced thereinto by the lithium acetate method. As a selection medium, the pre-culture medium shown in Table 1 and Table 2 was used. Thus, Yeast 1 of Invention was obtained. In the same manner, Yeasts 2 to 38 of Invention each comprising the response element according to the present invention and Control yeasts 1 to 9 shown in Table 3 were prepared.

TABLE-US-00003 TABLE 3 Response element Response Transcription Coupling type element unit factor factor Yeast 1 ofInvention SEQ ID NO: 2 1 DampaMET DampaSRC Yeast 2 ofInvention SEQ ID NO: 2 2 DampaMET DampaSRC Yeast 3 of Invention SEQ ID NO: 2 3 DampaMET DampaSRC Yeast 4 ofInvention SEQ ID NO: 2 4 DampaMET DampaSRC Yeast 5 ofInvention SEQ ID NO: 2 5 DampaMET DampaSRC Yeast 6 ofInvention SEQ ID NO: 5 3 DampaMET DampaSRC Yeast 7 ofInvention SEQ ID NO: 8 3 DampaMET DampaSRC Yeast 8 ofInvention SEQ ID NO: 2 4 DmMET DmTai Yeast 9 ofInvention SEQ ID NO: 34 1 DampaMET DampaSRC Yeast 10 ofInvention SEQ ID NO: 34 2 DampaMET DampaSRC Yeast 11 ofInvention SEQ ID NO: 34 3 DampaMET DampaSRC Yeast 12 ofInvention SEQ ID NO: 34 4 DampaMET DampaSRC Yeast 13 ofInvention SEQ ID NO: 34 5 DampaMET DampaSRC Yeast 14 ofInvention SEQ ID NO: 8 1 DampaMET DampaSRC Yeast 15 ofInvention SEQ ID NO: 8 2 DampaMET DampaSRC Yeast 16 ofInvention SEQ ID NO: 8 4 DampaMET DampaSRC Yeast 17 ofInvention SEQ ID NO: 37 1 DampaMET DampaSRC Yeast 18 ofInvention SEQ ID NO: 37 2 DampaMET DampaSRC Yeast 19 ofInvention SEQ ID NO: 37 3 DampaMET DampaSRC Yeast 20 ofInvention SEQ ID NO: 37 4 DampaMET DampaSRC Yeast 21 ofInvention SEQ ID NO: 40 1 DampaMET DampaSRC Yeast 22 ofInvention SEQ ID NO: 40 2 DampaMET DampaSRC Yeast 23 ofInvention SEQ ID NO: 40 3 DampaMET DampaSRC Yeast 24 ofInvention SEQ ID NO: 40 4 DampaMET DampaSRC Yeast 25 ofInvention SEQ ID NO: 43 1 DampaMET DampaSRC Yeast 26 ofInvention SEQ ID NO: 43 2 DampaMET DampaSRC Yeast 27 ofInvention SEQ ID NO: 43 3 DampaMET DampaSRC Yeast 28 ofInvention SEQ ID NO: 43 4 DampaMET DampaSRC Yeast 29 ofInvention SEQ ID NO: 46 1 DampaMET DampaSRC Yeast 30 ofInvention SEQ ID NO: 46 2 DampaMET DampaSRC Yeast 31 ofInvention SEQ ID NO: 46 3 DampaMET DampaSRC Yeast 32 ofInvention SEQ ID NO: 49 1 DampaMET DampaSRC Yeast 33 ofInvention SEQ ID NO: 49 2 DampaMET DampaSRC Yeast 34 ofInvention SEQ ID NO: 49 3 DampaMET DampaSRC Yeast 35 ofInvention SEQ ID NO: 49 4 DampaMET DampaSRC Yeast 36 ofInvention SEQ ID NO: 52 1 DampaMET DampaSRC Yeast 37 ofInvention SEQ ID NO: 52 2 DampaMET DampaSRC Yeast 38 ofInvention SEQ ID NO: 52 3 DampaMET DampaSRC Control yeast 1 cacgcggcggcacgtg 1 DampaMET DampaSRC Control yeast 2 cacgcggcggcacgtg 2 DampaMET DampaSRC Control yeast 3 cacgcggcggcacgtg 3 DampaMET DampaSRC Control yeast 4 cacgcggcggcacgtg 4 DampaMET DampaSRC Control yeast 5 DmJHRR 3 DampaMET DmTai Control yeast 6 DmJHRR 3 DmMET DmTai Control yeast 7 SEQ ID NO: 25 4 DampaMET DampaSRC Control yeast 8 SEQ ID NO: 28 4 DampaMET DampaSRC Control yeast 9 SEQ ID NO: 31 4 DampaMET DampaSRC

[0093] The test examples described below were performed using the yeast transformants.

[Test Example 1] Assay of Reporter Activity of Yeast Transformant to Juvenile Hormones

[0094] With the use of the Yeast 1 of Invention, reporter activity to methyl farnesoate as a juvenile hormone substance was measured. At the outset, the Yeast 1 of Invention was subjected to pre-culture at 30° C. for 18 hours using the pre-culture medium shown in Table 1 until the turbidity (O.D. 595) of the pre-culture medium reached approximately 0.1. The solution of the pre-cultured cells (10 μl) and 1 μl of the solution of methyl farnesoate diluted to various concentrations with dimethyl sulfoxide (DMSO) were mixed with 90 μl of the main culture solution shown in Table 4 in a 96-well plate, and the mixture was subjected to static culture at 30° C. for 18 hours to prepare a reaction solution. The resultant was designated as a test group.

TABLE-US-00004 TABLE 4 Composition of main culture medium Components Amount Yeast nitrogen base w/o amino acids and ammonium sulfate 1.7 g (NH.sub.4).sub.2SO.sub.4 5.0 g Drop-out powder (Table 2) 1.3 g 5M NaOH 500 μl D(+)-galactose 10 g Water 11

[0095] A reaction solution prepared with the addition of 1 μl of DMSO instead of a diluted methyl farnesoate solution was designated as a control group. Fractions of 10 μl each were collected from the reaction solutions and dispensed into each well of another 96-well plate. Thereafter, 100 μl of an assay reagent comprising a lytic solution (Z buffer: 60 mM Na.sub.2HPO.sub.4, 40 mM NaH.sub.2PO.sub.4, 1 mM MgCl.sub.2, 10 mM KCl, 2 mM dithiothreitol, 0.20% N-lauroylsarcosine sodium salt) mixed with 1 mg/ml ONPG (orthonitrophenylgalactopyranoside) was dispensed into each well, and the reaction was allowed to proceed at 37° C. for 30 minutes. Thereafter, the absorbance (O.D. 405) and the turbidity (O.D. 595) of the reaction solutions were assayed at the wavelength of 405 nm using a microplate reader (tradename: iMark microplate reader, Bio-Rad Laboratories). The increase of induction was calculated in accordance with the following formula.

[00001]$\mathrm{Increase}\mathrm{in}\mathrm{induction}=\frac{O.D\text{.405}\mathrm{of}\mathrm{Test}\mathrm{group}}{O.D\text{.595}\mathrm{of}\mathrm{Test}\mathrm{group}}-\frac{O.D\text{.405}\mathrm{of}\mathrm{Control}\mathrm{group}}{O.D\text{.595}\mathrm{of}\mathrm{Control}\mathrm{group}}$

[0096] The increase of induction is described in Ito-Harashima S et al., FEBS Open Bio., 7 (7): pp. 995-1008, 2017. When the increase of induction is positive, the transformant is determined to have reporter activity to the substance tested. The test was performed once or repeated 3 times, 5 times, or 15 times, and the average increase of induction was calculated. The control yeast 1 was treated in the same manner and reporter activity was compared. The results are shown in Table 5.

TABLE-US-00005 TABLE 5 Amount of juvenile hormone (methyl farnesoate) treated Tested yeast transformant 100 pM 1 nM Yeast 1 of Invention 0.10 0.08 Control yeast 1 0.01 0.00

[0097] As shown in Table 5, reporter gene expression is induced in the presence of methyl farnesoate, which is a juvenile hormone, in the Yeast 1 of Invention comprising the response element. In the control yeast 1, in contrast, substantially no reporter activity was detected.

[Test Example 2] Assay of Reporter Activity of Yeast Transformant to Juvenile Hormones

[0098] In the same manner as in Test Example 1, the Yeasts 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 of Invention were subjected to assays of reporter activity to methyl farnesoate. The test was performed in accordance with Test Example 1, and reporter activity of the yeasts indicated above was compared with that of the control yeasts 2, 3, and 4. The results are shown in Table 6 and Table 7.

TABLE-US-00006 TABLE 6 Amount of juvenile hormone (methyl famesoate) treated Tested yeast transformant 10 nM 100 nM 1 μM 10 μM 100 μM Yeast 2 of Invention 0.54 1.26 1.45 1.64 1.57 Yeast 3 of Invention 2.05 4.35 4.45 4.07 4.29 Yeast 4 of Invention 5.13 7.55 7.12 7.44 7.47 Yeast 5 of Invention 1.33 3.29 3.72 3.76 4.05 Yeast 6 of Invention 0.33 1.19 1.18 1.33 1.33 Yeast 7 of Invention 0.06 0.26 0.32 0.36 0.34 Control yeast 2 −0.29 −0.27 −0.34 −0.21 −0.14 Control yeast 3 −0.12 −0.21 −0.27 −0.17 −0.17 Control yeast 4 −0.84 −1.49 −1.82 −1.05 −2.06

TABLE-US-00007 TABLE 7 Amount of juvenile hormone (methyl farnesoate) treated Tested yeast transformant 100 nM 10 μM Yeast 9 of Invention 0.19 0.16 Yeast 10 of Invention 0.08 0.20 Yeast 11 of Invention 0.22 0.24 Yeast 12 of Invention 1.43 2.55 Yeast 13 of Invention — 0.09 Yeast 14 of Invention — 0.09 Yeast 15 ofInvention 0.09 0.16 Yeast 16 ofInvention 0.65 1.32 Yeast 17 ofInvention 0.06 0.09 Yeast 18 ofInvention 0.10 — Yeast 19 ofInvention 1.53 2.27 Yeast 20 ofInvention 1.55 1.85

[0099] As shown in Table 6 and Table 7, it is apparent that potent expression of the reporter gene is induced in the presence of ajuvenile hormone (methyl farnesoate) in the Yeasts 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 of Invention. In contrast, the increase of induction was negative in the control yeasts 2, 3, and 4 in any amount of treatment, and such control yeasts did not show any reporter activity. The results demonstrate that use of a nucleotide sequence comprising a total of 20 to 60 nucleotides including the nucleotide sequence shown in SEQ ID NO: 1 as a response element would enable evaluation of reporter activity to methyl farnesoate, which is a juvenile hormone, with high sensitivity.

[Test Example 3] Influence of Core Sequence on Reporter Activity of Yeast Transformant

[0100] With the use of the control yeast 7 comprising a different nucleotide sequence of the C-box (CACGCG) in the response element, the control yeast 8 comprising a different nucleotide sequence of the E-box (CACGTG), and the control yeast 9 comprising different nucleotide sequences of the C-box and the E-box from the relevant nucleotide sequences of the Yeast 4 of Invention, the influence of a core sequence on reporter activity to methyl farnesoate was evaluated. The test was performed in accordance with Test Example 1. The results are shown in Table 8.

TABLE-US-00008 TABLE 8 Amount of juvenile hormone (methyl farnesoate) treated Tested yeast transformant 100 nM 10 μM Yeast 4 of Invention 0.42 0.51 Control yeast 7 −0.42 −0.12 Control yeast 8 0.00 −0.02 Control yeast 9 −0.42 −0.12

[0101] As shown in Table 8, high reporter activity to methyl farnesoate was induced in the Yeast 4 of Invention. In the control yeasts 7, 8, and 9 each comprising different nucleotide sequences in the C-box and/or the E-box, in contrast, reporter activity was lowered to a significant extent, compared to the Yeast 4 of Invention.

[Test Example 4] Assay of Reporter Activity of Yeast Transformant to Sex Hormones and Chemical Substances Suspected of Having Endocrine Disrupting Actions

[0102] With the use of the Yeast 4 and the Yeast 8 of the present Invention, reporter activity to sex hormones; i.e., 17β-estradiol and testosterone, insect juvenile hormones; i.e., juvenile hormone III, and chemical substances suspected of having endocrine disrupting actions, such as 4-nonyl phenol, bisphenol A, 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT), 2,2-bis(4-chlorophenyl)-1,1-dichloroethylene (DDE), and dieldrin, was assayed. The test was performed in accordance with Test Example 1. All the test substances were treated at 10 μM. For comparison, reporter activity to compounds was assayed using the control yeast 5 and the control yeast 6 comprising a known juvenile hormone receptor response element (i.e., the JH response element of Drosophila melanogaster) integrated therein. The results are shown in Table 9.

TABLE-US-00009 TABLE 9 Tested yeast Increase in induction transformant 17β-estradiol Testosterone Juvenile hormone III 4-nonyl phenol Bisphenol A DDT DDE Dieldrin Yeast 4 of Invention 1.97 1.78 6.20 2.16 1.14 0.65 2.15 3.85 Yeast 8 of Invention 1.01 0.98 1.45 1.81 0.52 0.37 1.23 0.89 Control yeast 5 −0.14 −0.02 0.41 0.74 −0.38 −0.07 −0.08 0.00 Control yeast 6 −0.29 −0.22 0.58 3.05 −0.23 −0.24 −0.19 −0.02 Concentration (10 μM)

[0103] As shown in Table 9, reporter gene expression was induced in the Yeast 4 and the Yeast 8 of Invention, regardless of endocrine disrupting chemical types. Thus, the response element of the present invention was found to be responsive to all the tested endocrine disrupting chemicals. The Yeast 8 of Invention is a transformant having a transcription factor of Drosophila melanogaster (DmMET). The response element of the present invention was found to be responsive to, in addition to a transcription factor of Crustacea, a transcription factor of an insect; i.e., Drosophila melanogaster. In contrast, reporter gene expression was induced only in systems using juvenile hormone III and 4-nonyl phenol in the control yeast 5 and the control yeast 6 each having a juvenile hormone response element of Drosophila melanogaster (DmJHRR), and DmJHRR did not show responsiveness to other endocrine disrupting chemicals. The results demonstrate that reporter assays involving the use of the response element of the present invention enable detection of endocrine disrupting chemicals exerting various actions.

[Test Example 5] Assay of Reporter Activity to Antagonist Substance for Juvenile Hormone Receptors

[0104] With the use of the yeast 4 of the present invention, antagonist activity of juvenile hormone receptors to endrin, aldrin, and dieldrin was evaluated at 10 μM. The test was performed in accordance with Test Example 1, except that the test was performed in the presence of 10 nM methyl farnesoate. The results are shown in Table 10.

TABLE-US-00010 TABLE 10 Increase in induction Tested yeast transformant Endrin Aldrin Dieldrin Yeast 4 of Invention −0.03 −9.02 −12.46 0: Group treated with 10 nM methyl farnesoate Concentration (10 μM)

[0105] As shown in Table 10, the Yeast 4 of Invention was found to suppress an increase in the reporter activity caused by methyl farnesoate on all the tested substances. The results demonstrate that the use of a transformant comprising the response element of the present invention integrated thereinto enables detection of endocrine disrupting chemicals exerting antagonist activity.

[Test Example 6] Assay of Reporter Activity of Yeast Transformant to Active Ingredients of Conventional Agricultural Chemicals

[0106] In accordance with Test Example 1, endocrine disrupting actions on active ingredients of conventional agricultural chemicals; i.e., mepanipyrim, benthiavalicarb isopropyl, pyribencarb, pyroxasulfone, and fenquinotrione, were assayed using the Yeast 4 of Invention. Active ingredients of conventional agricultural chemicals were tested at 100 μM. All the tested active ingredients of conventional agricultural chemicals are known to exert no endocrine disrupting actions. The results are shown in Table 11.

TABLE-US-00011 TABLE 11 Tested yeast Increase in induction transformant Mepanipyrim Benthiavalicarb isopropyl Pyribencarb Pyroxasulfone Fenquinotrione Yeast 4 of −0.76 −0.37 −0.24 −0.50 −0.24 Invention Concentration (100 μM)

[0107] As shown in Table 11, reporter gene expression was not induced in the transformant comprising the response element of the present invention on all the active ingredients of conventional agricultural chemicals at 100 μM. The results demonstrate that a transformant comprising a nucleic acid encoding a receptor for endocrine disrupting chemicals having transcription factor activity, the response element according to the present invention, and a reporter gene introduced thereinto enables selective detection of endocrine disrupting chemicals.

[Test Example 7] Influence of Peripheral and Linker Regions of Core Sequence on Reporter Activity of Yeast Transformant

[0108] The influence of the core sequence on reporter activity to methyl farnesoate was evaluated using the Yeasts 21, 22, 23, and 24 of Invention comprising a nucleotide sequence derived from the nucleotide sequence shown in SEQ ID NO: 2 by substitution of 3 nucleotides outside the 5′ terminus of the C-box (CACGCG) in the response element; the Yeasts 25, 26, 27, and 28 of Invention comprising a nucleotide sequence derived from the nucleotide sequence shown in SEQ ID NO: 2 by substitution of 3 nucleotides outside the 3′ terminus of the E-box (CACGTG) in the response element; the Yeasts 29, 30, and 31 of Invention comprising a nucleotide sequence derived from the nucleotide sequence shown in SEQ ID NO: 2 by substitution of 3 nucleotides outside the 5′ terminus of the C-box (CACGCG) and 3 nucleotides outside the 3′ terminus of the E-box (CACGTG) in the response element; the Yeasts 32, 33, 34, and 35 of Invention each comprising a different nucleotide sequence in a region between the C-box and the E-box (a linker region); and the Yeasts 36, 37, and 38 of Invention comprising a nucleotide sequence derived from the nucleotide sequence shown in SEQ ID NO: 2 by substitution of 3 nucleotides outside the 5′ terminus of the C-box (CACGCG), 3 nucleotides outside the 3′ terminus of the E-box (CACGTG), and a region between the C-box and the E-box (a linker region). Methyl farnesoate was treated at 100 nM and 10 μM. The test was performed in accordance with Test Example 1. The results are shown in Table 12.

TABLE-US-00012 TABLE 12 Amount of juvenile hormone (methyl farnesoate) treated Tested yeast transformant 100 nM 10 μM Yeast 21 of Invention 0.20 0.19 Yeast 22 of Invention 0.10 0.20 Yeast 23 of Invention 0.49 1.09 Yeast 24 of Invention 0.93 1.66 Yeast 25 of Invention 0.06 0.15 Yeast 26 of Invention 0.35 0.38 Yeast 27 of Invention 0.03 0.64 Yeast 28 of Invention 1.87 2.51 Yeast 29 of Invention 0.07 0.07 Yeast 30 of Invention 0.16 0.07 Yeast 31 of Invention 0.09 0.23 Yeast 32 of Invention 0.11 0.31 Yeast 33 of Invention 0.42 0.22 Yeast 34 of Invention 0.66 2.15 Yeast 35 of Invention 1.44 1.05 Yeast 36 of Invention 0.06 1.38 Yeast 37 of Invention 0.17 0.13 Yeast 38 of Invention 0.09 0.04

[0109] As shown in Table 12, it is apparent that reporter gene expression is more potently induced in the presence of juvenile hormone (methyl farnesoate) in the Yeasts 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38 of Invention. The results demonstrate that a nucleotide sequence consisting of a total of 20 to 60 nucleotides would enable evaluation of reporter activity to methyl farnesoate, which is a juvenile hormone, with high sensitivity, even if 4 nucleotides in a region (a linker region) between the C-box (CACGCG) and the E-box (CACGCG) are kmkk (wherein k indicates G or t; and m indicates A or C) or, for example, TATT other than GCGG. Even if a nucleotide sequence in the vicinity of the nucleotide sequence shown in SEQ ID NO: 1 is different from the nucleotide sequence shown in SEQ ID NO: 2, in addition, reporter activity to juvenile hormone-like substances can also be evaluated with high sensitivity.