JP2004248604A5 - - Google Patents

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出典：酵母ラボマニュアル 酵母分子細胞生物学実験法 シュプリンガー・フェアラーク東京株式会社 1998 pp.5-6
【００１０】
本発明では、微生物として酵母Saccharomyces cerevisiae W303株 (American Type Culture Collection ATCC 2001239) にLEU2遺伝子を挿入しさらにURA3遺伝子を有するプラスミドで形質転換したものを用いる場合、SD培地にアデニン、ヒスチジン、トリプトファンを添加した培地を基本とし、これに種々のアミノ酸を添加するのが、好ましい。
この態様における、本発明に用いられる培地の具体例を以下に示す。
ａ）合成選択培地にアミノ酸を添加する例
・SD培地＋グルコース＋アミノ酸（アデニン、L-ヒスチジン、L-トリプトファン）
・SD培地＋グルコース＋アミノ酸（アデニン、L-ヒスチジン、L-トリプトファン、ウラシル）
・SD培地＋グルコース＋アミノ酸（アデニン、L-ヒスチジン、L-トリプトファン、L-アルギニン）
・SD培地＋グルコース＋アミノ酸（アデニン、L-ヒスチジン、L-トリプトファン、L-アスパラギン酸）
・SD培地＋グルコース＋アミノ酸（アデニン、L-ヒスチジン、L-トリプトファン、L-グルタミン酸）
・SD培地＋グルコース＋アミノ酸（アデニン、L-ヒスチジン、L-トリプトファン、L-ロイシン）
・SD培地＋グルコース＋アミノ酸（アデニン、L-ヒスチジン、L-トリプトファン、L-リシン）
・SD培地＋グルコース＋アミノ酸（アデニン、L-ヒスチジン、L-トリプトファン、L-メチオニン）
・SD培地＋グルコース＋アミノ酸（アデニン、L-ヒスチジン、L-トリプトファン、L-フェニルアラニン）
・SD培地＋グルコース＋アミノ酸（アデニン、L-ヒスチジン、L-トリプトファン、L-セリン）
・SD培地＋グルコース＋アミノ酸（アデニン、L-ヒスチジン、L-トリプトファン、L-トレオニン）
・SD培地＋グルコース＋アミノ酸（アデニン、L-ヒスチジン、L-トリプトファン、L-トリプトファン）
・SD培地＋グルコース＋アミノ酸（アデニン、L-ヒスチジン、L-トリプトファン、L-チロシン）
・SD培地＋グルコース＋アミノ酸（アデニン、L-ヒスチジン、L-トリプトファン、L-バリン）
ｂ）SD培地の糖組成としてグルコースの代わりにガラクトース、もしくはラフィノースを加えたもの
・SD培地＋ガラクトース＋アミノ酸（アデニン、L-ヒスチジン、L-トリプトファン）
・SD培地＋ラフィノース＋アミノ酸（アデニン、L-ヒスチジン、L-トリプトファン）
【００１１】
ｃ） 富栄養培地
・YPD培地
【００１２】
データベース作成に当たり、上記組成の培地のなかから、好ましくは５から１０個程度を選択する。
【００１３】
データベース作成に用いる培養培地に添加する「毒性物質」は、農薬、医薬品、染料、塗料、接着剤等の産業用途を有し環境に放出され可能性の高い化学物質および消毒・焼却の過程により非意図的に生成される化学物質であり、毒性を有することが懸念されるものである。これらの物質の毒性作用として、内分泌撹乱物作用、変異原性、発がん性、遺伝毒性、生態毒性、免疫毒性、細胞機能障害毒性等が知られている。
【００１４】
具体的な「毒性物質」としては例えば、表２に示すものが挙げられる。
【表２】

【表３】

【００１５】
他の具体例として、以下の表３に示すものが挙げられる。
【表４】

【表５】

【表６】

【表７】

【表８】

【表９】

【表１０】

【表１１】

【表１２】

【表１３】

【００１６】
データベース作成の際には、添加に際し選択する毒性物質は多ければ多い方が良いが、好ましくは２０から３０個程度、上記毒性物質のなかから選択する。
用いる毒性物質の濃度は、用いる微生物の生育を阻害するが、死滅に至らない濃度であり、通常個々の毒性物質のＩＣ５０値を用いる。生物を用いた実験はその結果がばらつく。よって、ＩＣ５０値を求める場合も３回以上の実験を行いその平均値を算出する。そこで、生物由来の実験誤差を無くする為に、データーベース作成時に細胞の生育に影響を与える濃度条件を決定するという方法が考えられる。
微生物が酵母の場合、毒性物質の濃度は次のようにして決定することもできる。毒性物質の希釈系列を作成し、初期の対数増殖期にある細胞（OD660=0.2〜0.5）に負荷し、さらに25℃で４時間培養する。この時、毒性物質を負荷しない細胞も同様に培養し対照区とする。２時間後、各濃度の毒性物質を添加したものおよび対照区の培養液の一部を取りYPD寒天培地に播種し、２日後にCFUを測定する（毒性物質負荷試料のCFUをCFU-chem、および対照区の試料のCFUをCFU-contと呼ぶ）。試料の残りはデータベース作成用の計測に供し、事後に決定した最適条件のもののみをデータとして用いる。
毒性物質添加直前に培養液の一部を取りYPD寒天培地に播種する。同様に、２時間後に各濃度の化学物質を添加したものおよび対照区の培養液の一部を取りYPD寒天培地に播種し、２日後にCFUを測定する（化学物質負荷直前の試料のCFUをCFU-before、毒性物質負荷試料のCFUをCFU-chem、および対照区の試料のCFUをCFU-contと呼ぶ）。酵母の倍加時間（doubling time）は2時間程度なので、CFU-contは通常CFU-beforeの２倍程度になる。異なる濃度の毒性物質を負荷した場合、細胞の増殖に影響を与える程度のCFU、すなわちCFU-contの50%以上の濃度、望ましくは75.5%に近くなる濃度を毒性物質の濃度として選択する（細胞が死滅する濃度ではCFUは0%であり、生育に影響を与えない濃度ではCFUは対照区と等しく100%となる）。
【００１７】
データベース作成に用いる「培養培地」の調製は当業者に周知である。例えば、上記のとおり種々選択した組成の溶液を、121℃、20分間オートクレーブすることにより滅菌する。アミノ酸は個々に高濃度の水溶液を作成し滅菌したものを用意し、必要に応じて最終濃度が上記表１となるように添加する。毒性物質は水またはジメチルスルホキシドに溶解し、最終濃度は前述の様に希釈系列を作成して実験を行う、もしくは事前に測定したIC50濃度になるように培地に添加する。
【００１８】
データベース作成に用いる「微生物の培養」は通常、４時間、２５℃にて行う。
【００１９】
データベース作成に用いる「微生物」は、天然に存在する野生株または野生型株、例えばヒト、マウスその他の哺乳類由来の動物細胞および動物細胞の樹立株、これまでバイオアッセイに用いられている魚類、線虫等の細胞、昆虫細胞、酵母等の真菌細胞、および大腸菌等の細菌細胞の何れであっても良い。ここに、野生株は天然に存在する組換えを行っていない微生物である。野生型株は注目する遺伝子に手をつけていない微生物であり、例えばW303は栄養要求性等について組換えされているが、それ以上の組換えはされていない。樹立株は動物細胞で継代培養できるもの、例えばガン細胞などの組換えが施されていないものも含む。
天然に存在する細胞もしくは樹立された細胞株を形質転換せずに用いた場合、データベース作成に用いる「微生物が示す応答」は毒性物質の存在下に変動する遺伝子のｍＲＮＡレベルである。ｍＲＮＡレベルは、ノーザンブロット法（緒方宣邦、野島博：遺伝子工学キーワードブック 改定第２版、羊土社、2000, pp299-301）、逆転写ＰＣＲ法（ＲＴ−ＰＣＲ）（中別府雄作、他：細胞工学別冊 Tipsシリーズ 改定PCR Tips, 秀潤社、1999, pp25-43）等によって測定できる。
ここに、「毒性物質の存在下に変動する遺伝子」は、酵母細胞の場合、公共のデータベース（例えば、ドイツのＭＩＰＳ：Munich Information Center for Protein Sequence、米国のＳＧＤ：Saccharomyces Genome Database）に開示されている酵母遺伝子の中から適宜選択し、これらはインターネットを介して知ることができる。好ましい遺伝子は、ミトコンドリアタンパク質、遺伝子修復系タンパク質、エネルギー系タンパク質、トランスポート促進タンパク質、ストレスタンパク質、代謝系タンパク質、脱毒性タンパク質および機能未知の遺伝子である。例えば、YKL071W、YCR102C、YOR382W、YLL057C、YLR303W等である。
【００２０】
データベース作成に用いる「微生物」はまた、上記天然に存在する細胞の組換え体、即ち形質転換体とすることができる。本発明に用いる形質転換体は、上記「毒性物質の存在下に変動する遺伝子」のプロモーターのポリヌクレオチド配列に、マーカータンパク質をコードするポリヌクレオチドを作動可能に連結して得られるポリヌクレオチド構築物を含むベクターを上記天然に存在する細胞に導入することにより調製できる。プロモーターにタンパク質をコードするポリヌクレオチドを作動可能に連結する方法は周知である（例えばR.W.オールド、S.B.プリムローズ 遺伝子操作の原理 原書第５版, 培風館, pp138-165, pp.234-263, 2000を参照）。
【００２１】
プロモーターの配列に関連し、例えば酵母を用いる場合、酵母の全DNA配列は明らかにされているので、公共のデータベース（SCPD：The Promoter Database of Saccharomyces cerevisiae）を用いることにより、プロモーターを含む酵母遺伝子配列を得ることができる。
マーカータンパク質の例としてはGFP（Green Fluorescence Protein）（Heim, R., Cubitt, A. B. and Tsien, R. Y.（1995） Nature 373, 663-664；Heim, R., Prasher DC. and Tsien, R. Y.（1994） Proc. Natl. Acad. Sci., 91, 12501-12504；Warg, S. and Hazerigg, T.（1994）Nature 639, 400-403；Youvan, D.C. and Michel-Beyerle, M.E.（1996）Nature Biotechnology 14 1219-1220；Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. and Prasher, D.C.（1994）Science 263, 802-805）、β-ガラクトシダーゼ（Canestro C, Albalat R, Escriva H, Gonzalez-Duarte R. Endogenous beta-galactosidase activity in amphioxus: a useful histochemical marker for the digestive system.Dev Genes Evol 2001 Mar 211(3):154-6）、ルシフェラーゼ（Arch Toxicol 2002 Jun;76(5-6):257-61、Estrogenic activity of UV filters determined by an in vitro reporter gene assay and an in vivo transgenic zebrafish assay.Schreurs R, Lanser P, Seinen W, Van Der Burg B.)、及びアセチルトランスフェラーゼ（J Recept Signal Transduct Res 2001 Feb;21(1):71-84, A simplified method for large scale quantification of ranscriptional activity and its use in studies of steroids and steroid receptors. Zhang S, Lu J, Iyama K, Lo SC, Danielsen M.)等が挙げられる。
【００２２】
ベクターの調製は、例えば酵母細胞の場合、酵母において複製可能なプラスミドを選択し、これにマーカータンパク質をコードするポリヌクレオチドを挿入し、次いでプロモーター配列を含むポリヌクレオチドをマーカー遺伝子の上流部分に挿入することにより行うことができる。
形質転換体は周知の手法により調製できる。例えば、酵母細胞を形質転換する方法は、Kaiser C, Michaelis S, Mitchell A: A Cold Spring Harbor Laboratory Course Manual 1994 edition (Cold Spring Harbor Laboratory Press) pp.133-134,1994に記載されている。
形質転換体を用いた場合、本発明のデータベース作成に用いる「微生物が示す応答」はマーカータンパク質の発現レベルである。マーカータンパク質の発現レベルは、形質転換体を粉砕しタンパク質抽出液を得、この液中のマーカータンパク質量を測定することにより得られる。例えばマーカータンパク質がＧＦＰの場合、タンパク質抽出液の蛍光量を蛍光分光光度計により計測する。また、マーカータンパク質の発現レベルは、形質転換体を粉砕しなくても直接、蛍光顕微鏡、レーザー顕微鏡による画像処理、フローサイトメトリーによる計測、エバネッセント光による計測、さらに比色計などを用いた検出方法により、細胞の蛍光（GFP）もしくは発色（β-gal）を測定できる場合もある。
【００２３】
好ましい形質転換体は、酵母遺伝子のプロモーターをマーカータンパク質をコードするポリヌクレオチドに作動可能に連結したポリヌクレオチドを含むベクターによって形質転換されている酵母、または染色体のORFをマーカータンパク質をコードするポリヌクレオチドで置換するように形質転換されている酵母であり、さらに好ましくは、その酵母遺伝子がミトコンドリアタンパク質、遺伝子修復系タンパク質、エネルギー系タンパク質、トランスポート促進タンパク質、ストレスタンパク質、代謝系タンパク質、脱毒性タンパク質および機能未知の遺伝子のなかから選ばれるものである。
【００２４】
データベース作成に用いる「微生物が示す応答の差異の指標化」を、マーカータンパク質としてGFPを用いた場合を例として以下に説明する。
毒性物質と接触後、マーカータンパク質の発現レベルを上記のようにして測定する。酵母細胞を生理食塩水で洗浄し、その後５％ホルマリンを含む生理食塩水で固定を行い、フローサイトメトリー（例えばEPICS XL：ベックマンコールター）を用いて一回の実験につき10000個の酵母細胞の蛍光を計測し、これらの細胞の蛍光強度の平均値を得る。対照区の蛍光強度の平均値との比を求め、例えばその比が１．５以下であれば「−」、１．５−２．０であれば「＋」、２以上であれば「＋＋」などと指標化する。
【００２５】
上記の通り、得られたデータは、表または電子情報化された形式としてまとめ、データベースとすることができる。
【００２６】
本発明は、上記のようにして作成された本発明データベースの使用方法であって、微生物を生育させる培養培地におけるアミノ酸組成および／または糖組成の違いにより、微生物が化学物質に対して示す応答の差異を指標に用いることを特徴とする化学物質を検出するための方法において、指標化された応答に対して照合する、該データベースの使用方法をも提供する。
【００２７】
２）化学物質の検出方法
本発明はさらに、微生物を生育させる培養培地におけるアミノ酸組成および／または糖組成の違いにより、微生物が化学物質に対して示す応答の差異を指標に用いることを特徴とする、化学物質を検出するための方法に関する。詳細には、被検試料中の化学物質を検出するための微生物を用いる方法であって、
（１）本発明のデータベースを作成するのに用いた培養培地におけるアミノ酸組成および糖組成と同じ組成を有する複数の培養培地にて、該被検試料の存在下、微生物を培養し、
（２）個々の培養培地における微生物が示す応答の差異をそれぞれ指標化し、
（３）指標化された応答の差異を、該データベースの指標と照合し、
それにより、該データベースにおける指標と同じ指標を示す該被検試料中の化学物質を検出する方法に関する。
【００２８】
本発明はまた、被検試料中の化学物質を検出するための微生物を用いる方法であって、
（１）種々のアミノ酸組成および種々の糖組成を有する培養培地それぞれに特定の毒性物質を添加した複数の培養培地を調製し、
（２）個々の得られた培養培地にて、該被検試料の存在下、微生物を一定期間培養し、
（３）個々の培養培地における微生物が示す応答の差異をそれぞれ指標化し、
（４）指標化された応答の差異を基準として、該被検試料中の化学物質を検出する、ことを特徴とする方法に関する。
【００２９】
本発明の化学物質の検出方法において「培養培地のアミノ酸組成および調製」、「毒性物質」、「微生物およびその培養」、「微生物が示す応答の差異およびその指標化」は、上記本発明のデータベース作成の説明と同様である。
【００３０】
上記の通り、本発明は、微生物の生育に用いる培養培地の組成の違いにより化学物質に対する微生物の応答が異なるという発見を基本とする。本発明の化学物質検出方法は、培養培地の組成を変えることにより、検出可能な化学物質の種類を増やすことが可能である。
【００３１】
【実施例】
以下に本発明を実施例によりさらに詳細に説明するが、本発明はこれら実施例に制限されるものではない。
【００３２】
参考例１
レポーター・ジーン・アッセイ
酵母遺伝子YBR072Wのプロモーター配列を含むポリヌクレオチド（SCPD：The Promoter Database of Saccharomyces cerevisiaeにて開示）をPCRにより増幅するためのプライマーを作成した。プライマーはプライマー設計用のソフトウェアであるOligo4.0-S，Sequencher_Iマッキントッシュ版を用いて設計し、アッパープライマーの塩基配列は、
GCAGTCAACGAGGAGCGAATCA （配列番号１）
であり、ロウワープライマーの塩基配列は、
GGGGTACCCCGTTAATTTGTTTAGTTTGTTTG （配列番号２）
とした。PCRはテンプレートとして酵母の染色体（Saccharomyces cerevisiae S288C, Cat.40802，Reserch Genetics, Inc.）を用い、試薬は市販のキット（KOD DNA Polymerase；コードKOD-101、Toyobo）を使用した。
使用するベクターは大腸菌と酵母の両方で複製されるYEp型シャトルベクターであるｐYES2（pYES2, Cat no:V825-20, Invitrogen Corporation, USA）（R.W.オールド、S.B.プリムローズ 遺伝子操作の原理 原書第５版, 培風館, pp.234-263, 2000））を用いた。また、マーカータンパク質ＧＦＰをコードするポリヌクレオチドはベクターpQBI 63（Cat no.54-0082, 和光純薬工業(株)）のGFPの部分（配列番号３）を用いた。まず、ｐYES2の多重クローニング部位（multiple cloning site）の中にＧＦＰのポリヌクレオチドを挿入したベクターを作成した。次いで、ｐYES2のGAL1プロモーターの部分を酵母遺伝子であるYBR072Wのプロモーター配列を含むポリヌクレオチドと置換し、目的とするプラスミドベクターを得た。ＧＦＰおよびプロモーター配列を含むポリヌクレオチドの挿入操作は、適当な制限酵素を選択して行った。
【００３３】
次に、このプラスミドベクターで酵母Saccharomyces cerevisiae W303（American Type Culture Collection ATCC 2001239）を形質転換した。形質転換の手順を以下に示す。
１）酵母細胞Saccharomyces cerevisiae W303をYPD培地200mlにてOD660が０．５になるまで振とう培養する。
２）集菌して５ｍｌのTE-bufferに懸濁する。
３）2.5Mのリチウムアセテイト250μLを添加する。
４）300μｌずつ分注し10μlの上記プラスミドベクターを添加し、３０℃３０分培養する。
５）700μｌの５０％PEG4000を添加し、３０℃６０分振とう培養する。
６）ヒートショック（４２℃、５分）後、急冷する。
７）１Mソルビトールで２回洗浄する。
８）最小栄養培地（SD培地に必要なアミノ酸（アデニン、ヒスチジン、トリプトファン）を加えたもの）で作成した寒天プレートに播種する。
【００３４】
形質転換の確認は選択培地（ＳＤ培地（Yeast nitrogen base without amino acids (Difco 0919-15)＋グルコース＋アミノ酸（アデニン、ヒスチジン、トリプトファン）により行った。選択培地の寒天プレートに生育したコロニーはさらに、アミノ酸の栄養要求性を確認した。
得られた形質転換体はＹＰＤ培地（酵母エキス１％、ポリペプトン２％、ブドウ糖２％）上にて４８時間まで安定に当該プラスミドを保持していることを確認した。
【００３５】
実施例１
化学物質負荷実験
参考例１にて調製した形質転換体を以下の表４に示す濃度の各化合物と、下記の培養条件下にて接触させる。
【表１４】
表４
酵母Ｗ３０３の生育能に対する化学物質の効果（ＩＣ５０）
化学物質 濃度
（１）２−アミノアントラセン 33.3 mM
（２）ベンゾ（ａ）ピレン 0.2 mM
（３）ビスフェノールＡ 0.4 mM
（４）フタル酸ジ（２−エチルヘキシル） 83.3 mM
（５）２，４−ジクロロフェノール 0.3 mM
（６）２，４−ジクロロフェノキシ酢酸 0.3 mM
（７）ホルムアルデヒド 0.2 mM
（８）塩化メチル水銀 0.2 μM
（９）４−ニトロキノリン−Ｎ−オキサイド 0.6 μM
（１０）ｐ−ノニルフェノール 10 μM
（１１）ペンタクロロフェノール 50 μM
（１２）亜ヒ酸ナトリウム 0.3 mM
（１３）チウラム 20 μM
（１４）トリブチルスズクロライド 0.4 μM
（１５）２，４，５−トリクロロフェノール 30 mM
（１６）Ｔｒｐ−Ｐ−２（酢酸塩） 0.2 mM
（１７）パラコート 16.7 mM
（１８）塩化カドミウム 40 μM
（１９）リンダン 6.7 mM
（２０）マラチオン 22.2 mM
（２１）マネブ 0.8 mM
（２２）塩化ニッケル（ＩＩ） 3.3 mM
（２３）重クロム酸カリウム（ＶＩ） 0.3 mM
（２４）トリフェニルスズクロライド（ＩＶ） 10 μM
（２５）フェノール 5.6 mM
（２６）チオベンカルブ 0.7 mM
（２７）ヘキサクロロフェン 30 μM
（２８）トリクロサン 730 μM
（２９）塩化水銀（ＩＩ） 50 μM
（３０）硫酸銅（ＩＩ） 3.3 mM
（３１）シアン化カリウム 16.7 mM
（３２）ジメチルスルホキシド 3.7 %

ＩＣ５０はYPD培地およびSD培地にて測定し、それらはほとんど等しいことを確認した。
【００３６】
培養条件
培養培地の組成
１．ＳＤ培地（Yeast nitrogen base without amino acids (Difco 0919-15)）＋グルコース＋アミノ酸（アデニン、ヒスチジン、トリプトファン）；
２．ＳＤ培地＋グルコース＋アミノ酸（アデニン、ヒスチジン、トリプトファン、ウラシル）
３．ＳＤ培地＋グルコース＋アミノ酸（アデニン、ヒスチジン、トリプトファン、ロイシン）
４．ＳＤ培地＋グルコース＋アミノ酸（アデニン、ヒスチジン、トリプトファン、メチオニン）
５．ＹＰＤ培地（酵母エキス１％、ポリペプトン２％、ブドウ糖２％）；および
６．ＳＤ培地＋ガラクトース＋アミノ酸（アデニン、ヒスチジン、トリプトファン）；
【００３７】
必要なアミノ酸を加えたSD培地10mlに凍結保存した形質転換体を白金耳で掻き取り、２５℃で振とう培養し、フルグロース（(定常状態まで増殖されそれ以上細胞数が増えない状態）させる。各培地１０mlにフルグロースさせた細胞けん濁液を50ul加え、２５℃で振とう培養し、OD660が0.2から0.5になった時点で化学物質を負荷する。化学物質負荷から４時間後、酵母細胞を生理食塩水で１回洗浄し、その後５％ホルマリンを含む生理食塩水で固定を行いフローサイトメトリー（EPICS XL：ベックマンコールター）を用いて一回の実験につき10000個の酵母細胞の蛍光を計測し、これらの細胞の蛍光強度の平均値を得た。対照区の蛍光強度の平均値との比を求め、指標化した。
得られた結果を表５に示す。表中、対照区に対し、蛍光強度が1.5倍以下を−、1.5倍以上2.0倍未満を＋、2.0倍以上を＋＋と表記した。
【００３８】
【表１５】

【表１６】

【００３９】
上記により、同じ形質転換体を用いても培養する培地の組成に応じて個々の化学物質に対する形質転換体の生育状態が変動することが証明された。本発明は、実施例記載のようにして作成される上記表５のデータを、データベースの一態様として提供する。
【００４０】
実施例２
被検試料中の化学物質検出
被検試料について、実施例１と同様、各組成を有する培養培地に添加し、同形質転換体を培養し、そしてその生育状態を上記基準に従って指標化することで指標のパターンを得る。それにより、同じパターンを示す化学物質が特定でき、被検試料中に存在し得る化学物質が推定される。
被検試料中に複数の化学物質が含まれる場合、毒性の種類の組合わせによって微生物が示す応答は相加もしくは相乗されることがある。しかし、実際的には同じ影響濃度の複数の化学物質という条件は非常に稀であるため、一番毒性の強い状態の化学物質が検出されると考えられる。
被検試料中に存在する化学物質の濃度が薄いと遺伝子発現は対照区と変化無いし、濃いと細胞が死滅するので遺伝子が発現しない。よって、IC50付近が本発明にとって好適な濃度である。
【００４１】
被検試料について好適な濃度を決定する方法
１．事前に濃度を決定しておく方法
被検試料が低濃度の場合は濃縮し、希釈系列を作り、高濃度の場合は希釈系列を作り、実験を行う。
希釈系列の試料を作成し、フルグロースの細胞を最小栄養培地で200倍に希釈した溶液に負荷する。２時間置きに４８時間までOD660を測定する。このとき、細胞の生育に影響を与えるが死滅しない濃度で、細胞の生育を５０％程度阻害する濃度を選択してIC50とする。
【００４２】
２．化学物質検出と同時に濃度を決定する方法
また、以下の様にしてＣＦＵを計測することで、好適な濃度を選ぶことができる。
化学物質添加直前に培養液の一部を取りYPD寒天培地に播種する。同様に、２時間後に各濃度の化学物質を添加したものおよび対照区の培養液の一部を取りYPD寒天培地に播種し、２日後にCFUを測定する（化学物質負荷直前の試料のCFUをCFU-before、化学物質負荷試料のCFUをCFU-chem、および対照区の試料のCFUをCFU-contと呼ぶ）。酵母のdoubling timeは2時間程度なので、CFU-contは通常CFU-beforeの２倍程度になる。異なる濃度の化学物質負荷した場合、細胞の増殖に影響を与える程度のCFU、すなわちCFU-contの50%以上95%以下の濃度、望ましくは75.5%に近くなる濃度を化学物質の濃度として選択する（細胞が死滅する濃度ではCFUは0%であり、生育に影響を与えない濃度ではCFUは対照区と等しく100%となる）。各濃度の化学物質を添加した細胞は、化学物質添加４時間後に生食塩水で１回洗浄し、その後５％ホルマリンを含む生理食塩水で固定を行い冷蔵保存しておき、上記の手順で適当な濃度を選択した後にデータを収集する。CFUを測定する替わりに、生細胞のみを染色する染色法を用いて、生細胞をカウントするという方法も考えられる。
【００４３】
【配列表】
SEQUENCE LISTING
<110> DAIKIN INDUSTRIES, LTD.

<120> Method for Detecting Chemical Substance with Microorganism

<130> 187131

<160> 3
<210> 1
<211> 22
<212> DNA
<213> Artificial Sequence

<400> 1
gcagtcaacg aggagcgaat ca 22

<210> 2
<211> 32
<212> DNA
<213> Artificial Sequence

<400> 2
ggggtacccc gttaatttgt ttagtttgtt tg 32

<210> 3
<211> 720
<212> DNA
<213> Aequorea victoria
<400> 3
atggctagca aaggagaaga actcttcact ggagttgtcc caattcttgt tgaattagat 60
ggtgatgtta acggccacaa gttctctgtc agtggagagg gtgaaggtga tgcaacatac 120
ggaaaactta ccctgaagtt catctgcact actggcaaac tgcctgttcc atggccaaca 180
ctagtcacta ctctgtgcta tggtgttcaa tgcttttcaa gatacccgga tcatatgaaa 240
cggcatgact ttttcaagag tgccatgccc gaaggttatg tacaggaaag gaccatcttc 300
ttcaaagatg acggcaacta caagacacgt gctgaagtca agtttgaagg tgataccctt 360
gttaatagaa tcgagttaaa aggtattgac ttcaaggaag atggcaacat tctgggacac 420
aaattggaat acaactataa ctcacacaat gtatacatca tggcagacaa acaaaagaat 480
ggaatcaaag tgaacttcaa gacccgccac aacattgaag atggaagcgt tcaactagca 540
gaccattatc aacaaaatac tccaattggc gatggccctg tccttttacc agacaaccat 600
tacctgtcca cacaatctgc cctttcgaaa gatcccaacg aaaagagaga ccacatggtc 660
cttcttgagt ttgtaacagc tgctgggatt acacatggca tggatgaact gtacaactga 720 [Document Name] Statement
[Title of the Invention] Using a microorganismChemistryMaterialdetectionMethod
[Claims]
    An amino acid composition and / or a sugar composition in a culture medium for growing a microorganism.Due to the differenceMicroorganismsFor chemical substancesIndicating responseDifferenceIs used as an index,ChemistrySubstancedetectionWay to do.
    (2) A method for detecting a chemical substance in a test sample,
(1) adding a test sample containing a chemical substance to be detected to each of culture media having various amino acid compositions and / or various sugar compositions,
(2) inoculating the microorganism in each culture medium of (1) and culturing for a certain period of time;
(3) The difference in the response of the microorganism to the chemical substance in the test sample is indexed by the difference in the composition of each culture medium.
(4) A method comprising detecting a chemical substance in the test sample based on the difference in the indexed response.
    (3) 3. The method of claim 1 or 2, wherein the database is checked for differences in indexed responses,
(1) preparing a plurality of culture media each having a specific toxic substance added to each of the culture media having various amino acid compositions and various sugar compositions;
(2) culturing microorganisms for a certain period in each obtained culture medium,
(3) indexing the difference in response of the microorganism in each culture medium,
(4) summarize the obtained indices as data,
The database created by.
    (4) 4. The database of claim 3, wherein the microorganism is yeast and the difference in response exhibited by the microorganism is a difference in mRNA levels of yeast genes.
    (5) The microorganism is a yeast which has been transformed by a vector comprising a polynucleotide operably linked to a polynucleotide encoding a marker protein, the promoter of the yeast gene, and the difference in response exhibited by the microorganism indicates the level of expression of the marker protein. 4. The database of claim 3, wherein the difference is:
    6. The database according to claim 4 or 5, wherein the yeast gene is selected from a mitochondrial protein, a gene repair system protein, an energy system protein, a transport promoting protein, a stress protein, a metabolic protein, a detoxicating protein, and a gene whose function is unknown. .
    7. 7. The method of claim 1 or 2, wherein the method compares the indexed response for differences.
    8. An apparatus for detecting a chemical substance, to which the database according to any one of claims 3 to 6 has been input.
    9. A method using a microorganism for detecting a chemical substance in a test sample,
(1) culturing a microorganism in a plurality of culture media having the same composition as the amino acid composition and the sugar composition in the culture medium used to create the database according to claim 3 in the presence of the test sample;
(2) indexing the difference in response of the microorganism in each culture medium,
(3) comparing the difference of the indexed response with the index of the database,
Thereby, a method of detecting a chemical substance in the test sample that shows the same index as the index in the database.
    10. 10. The method of claim 9, wherein the microorganism is yeast, and the difference in response exhibited by the microorganism is a difference in mRNA levels of yeast genes, and the database is as in claim 4.
    11. The microorganism is a yeast which has been transformed with a vector comprising a polynucleotide operably linked to a polynucleotide encoding a marker protein, the promoter of the yeast gene, and the difference in response exhibited by the microorganism indicates the level of expression of the marker protein. The method of claim 9, wherein the database is that of claim 5.
    12. The method according to claim 10 or 11, wherein the yeast gene is selected from a mitochondrial protein, a gene repair protein, an energy protein, a transport promoting protein, a stress protein, a metabolic protein, a detoxicating protein, and a gene whose function is unknown. .
    Claim 13 A method using a microorganism for detecting a chemical substance in a test sample,
(1) preparing a plurality of culture media each having a specific toxic substance added to each of the culture media having various amino acid compositions and various sugar compositions;
(2) culturing a microorganism for a certain period in each of the obtained culture media in the presence of the test sample,
(3) indexing the difference in response of the microorganism in each culture medium,
(4) A method characterized in that a chemical substance in the test sample is detected on the basis of the difference in the indexed response.
DETAILED DESCRIPTION OF THE INVENTION
      [0001]
    TECHNICAL FIELD OF THE INVENTION
  The present invention uses microorganismsChemistryMaterialdetectionAbout the method.
      [0002]
    [Prior art]
  To date, humans have created a vast number of chemicals, and new chemicals are being developed year by year. These chemicals are used in all aspects of modern life and help improve human life. On the other hand, some chemical substances are released into the environment at various stages, such as production, distribution, use, and disposal, and remain in the environment, through biological enrichment by the food chain, etc. Some have harmful effects on ecosystems, and environmental pollution has become a social problem. Therefore, there is a need to evaluate the effects of chemicals on humans and ecosystems.
  Conventionally, as an evaluation method, bioassays mainly using the inhibition of the growth of individuals, such as fish, daphnia, shellfish, and the like, and specific biological reactions have been performed. However, these methods can only determine the presence or absence of toxicity by environmental chemicals. As an approach to determine not only the presence or absence of toxicity from chemicals in the environment, but also the nature of the toxicity and which chemicals in the environment are attributable to the chemicals in the environment, several types of indicator chemicals at various concentrations and their presence There has been reported a technique that utilizes the relationship between the growth state of a plurality of types of indicator microorganisms below (Patent Document 1: JP-A-2001-238694). Other bioassays have been developed to detect specific toxicity indicators (endpoints). For example, the ames method, which is a gene mutation test in which 90% or more of known carcinogenic substances are positive (Non-Patent Document 1: “Bioassay for Physiologically Active Substances”, Kodansha, 1989, Third Edition, p.319), and For screening for endocrine disruptors, a test method for measuring gene transcription activity that occurs when in vivo hormones bind to receptors (Non-Patent Document 2: Yamasaki K, Takeyoshi M, Yakabe Y, Sawaki M, Imatanaka N, Takatsuki M .: Comparison of reporter gene assay and immature rat uterotrophic assay of twenty-three chemicals. Toxicology. 2002 Jan 15; 170 (1-2): 21-30.). However, these methods are effective for detecting a chemical substance having a specific toxicity, but cannot detect a chemical substance having a toxicity other than the toxicity index.
      [0003]
        [Patent Document 1]
          JP 2001-238694 A
        [Non-patent document 1]
          "Bioassay for Physiologically Active Substances", Kodansha, 1989, Third Edition, p.319
        [Non-patent document 2]
          Yamasaki K, et al., Toxicology. 2002 Jan 15; 170 (1-2): 21-30.
        [Non-Patent Document 3]
          Mao X, et al., Curr Microbiol 2002 Jul; 45 (1): 37-40
      [0004]
    [Problems to be solved by the invention]
  The present invention provides a new method for obtaining detailed information on chemicals in the environment, that is, for determining the nature of toxicity of chemicals in the environment and which chemicals in the environment cause the toxicity. Provide a simple way.
      [0005]
    [Means for Solving the Problems]
  The present inventor, in the evaluation of the toxicity of chemical substances using the reporter gene assay described in Steib P, Moran GP, et al. J Bacteriol 2001 May; 183 (9): 2859-65, etc. The present inventors have found that the response of a microorganism to a chemical substance varies by changing the composition of a culture medium used for growing the microorganism, and completed the present invention.
      [0006]
  That is, the present invention provides, as one embodiment, an amino acid composition and / or a sugar composition in a culture medium for growing a microorganism.Due to the differenceMicroorganismsFor chemical substancesIndicating responseDifferenceIs used as an index,ChemistrySubstancedetectionProvide a way to
  Of the present inventionChemistrymaterialdetectionIn the method, a database that is matched against the indexed microbial response for the test sample is important. The present invention, in another aspect, provides such a database and methods of using the same.
  Note that Mao X, Hu Y, Liang C, Lu C. Curr Microbiol 2002 Jul; 45 (1): 37-40 (Non-patent Document 3) shows that the methionine concentration in the medium is It is described that the effect was confirmed by a MET3 promoter reporter gene assay (βgal expression). The MET3 gene is known to be a gene encoding an enzyme that synthesizes methionine from extracellular sulfate (sulfate) (http://mips.gsf.de/proj/yeast/CYGD/db/ of MIPS). index.html). Therefore, Non-Patent Document 3 merely confirms the effect of methionine produced from the enzyme encoded by the MET3 gene on the expression of the MET3 gene itself. It depends on the enzyme product involved.
  On the other hand, the present invention is based on the discovery that the expression state of a gene for a chemical substance varies depending on the environment of the medium, that is, the presence or absence of amino acids and sugars.
      [0007]
    BEST MODE FOR CARRYING OUT THE INVENTION
  As described above, the present inventionChemistrymaterialdetectionThe method typically employs a database that is matched against a microbial response indexed for the test sample.
I. Database
  To create the database of the present invention,
(1) For each culture medium having various amino acid compositions and various sugar compositionsspecificToxic substances are preferably added to one or more amino acids and one or more sugars in a medium having the minimum amino acids required for growth, and added to the resulting medium.specificPrepare multiple culture media by adding toxic substances of
(2)IndividualThe microorganisms are cultured for a certain period in the obtained culture medium,
(3) Responses of microorganisms in individual culture mediaDifferenceAre indexed, respectively, and
(4) Collect the obtained indices as data.
  The database created in this way can be organized as a table shown in the following embodiment or in a form of electronic information. If the computerized database is input to a computer or the like, it can be easily used for collation with data obtained from a test sample. In this aspect, the invention also provides a device into which the database of the invention has been entered.
      [0008]
  "Amino acid composition and sugar composition in culture medium" used for database creation is the composition of a nutrient-rich general medium with a fast growth rate when a naturally occurring wild strain or a constant recombinant is used as a microorganism. . On the other hand, when a transient recombinant using a vector or a plasmid is used as a microorganism, a minimal nutrient medium is usually used as a selective pressure for retaining the plasmid (Gupta JC, Mukherjee KJ .: Biotechnol Bioeng). 2002 Jun 5; 78 (5): 475-88).
      [0009]
  The following may be mentioned as the medium used in the present invention:
1. YPD medium: Composition (in 1 L) Yeast extract 10 g 1% (w / v), polypeptone 20 g 2% (w / v), glucose 20 g 2% (w / v)
  This is a eutrophic medium and is used for normal culture. Amino acids are also abundant.
2. SD medium: Composition (in 1 L): yeast nitrogen base without amino acids (Difco.0919-15-3) 6.7 g 0.67% (w / v), glucose 20 g 2% (w / v)
  It is a synthetic selective medium and contains no amino acids. It is used to check the auxotrophy of strains and to select transformants. In the case of an auxotrophic strain, one or more amino acids may be added as necessary. It is sometimes called the minimum nutrient medium.
  In the present invention, amino acids of the types and concentrations shown in Table 1 were added to SD medium.acidOr other types of sugars, such as galactose and raffinose, in place of glucose in the SD medium described above, and amino acids having the types and concentrations shown in Table 1 are added.acidIs added.
[Table 1]

  OutScripture: Yeast Lab Manual Yeast Molecular Cell Biology Experimental Method Springer Verlag Tokyo Co., Ltd. 1998 pp.5-6
      [0010]
  In the present invention, when a microorganism obtained by inserting the LEU2 gene into the yeast Saccharomyces cerevisiae W303 strain (American Type Culture Collection ATCC 2001239) and further transforming it with a plasmid having the URA3 gene is used, adenine, histidine and tryptophan are added to the SD medium. It is preferable to add various amino acids to a basic medium based on the above.
  Specific examples of the medium used in the present invention in this embodiment are shown below.
a) Example of adding amino acid to synthetic selective medium
・ SD medium + glucose + amino acid (adenine, L-histidine, L-tryptophan)
・ SD medium + glucose + amino acid (adenine, L-histidine, L-tryptophan, uracil)
・ SD medium + glucose + amino acid (adenine, L-histidine, L-tryptophan, L-arginine)
・ SD medium + glucose + amino acid (adenine, L-histidine, L-tryptophan, L-aspartic acid)
・ SD medium + glucose + amino acid (adenine, L-histidine, L-tryptophan, L-glutamic acid)
・ SD medium + glucose + amino acid (adenine, L-histidine, L-tryptophan, L-leucine)
・ SD medium + glucose + amino acid (adenine, L-histidine, L-tryptophan, L-lysine)
・ SD medium + glucose + amino acid (adenine, L-histidine, L-tryptophan, L-methionine)
・ SD medium + glucose + amino acid (adenine, L-histidine, L-tryptophan, L-phenylalanine)
・ SD medium + glucose + amino acid (adenine, L-histidine, L-tryptophan, L-serine)
・ SD medium + glucose + amino acid (adenine, L-histidine, L-tryptophan, L-threonine)
・ SD medium + glucose + amino acid (adenine, L-histidine, L-tryptophan, L-tryptophan)
・ SD medium + glucose + amino acid (adenine, L-histidine, L-tryptophan, L-tyrosine)
・ SD medium + glucose + amino acid (adenine, L-histidine, L-tryptophan, L-valine)
b) Sugar composition of SD medium to which galactose or raffinose is added instead of glucose
・ SD medium + galactose + amino acid (adenine, L-histidine, L-tryptophan)
・ SD medium + raffinose + amino acid (adenine, L-histidine, L-tryptophan)
      [0011]
c) Rich nutrient medium
・ YPD medium
      [0012]
  In preparing the database, about 5 to 10 cells are preferably selected from the medium having the above composition.
      [0013]
  "Toxic substances" to be added to the culture medium used to create the database are non-toxic chemicals that have industrial uses such as agricultural chemicals, pharmaceuticals, dyes, paints, and adhesives, are likely to be released into the environment, and are disinfected and incinerated. It is a chemical substance that is intentionally generated and may be toxic. As toxic effects of these substances, endocrine disruptor effects, mutagenicity, carcinogenicity, genotoxicity, ecotoxicity, immunotoxicity, cytotoxicity and the like are known.
      [0014]
  Specific "toxic substances" include, for example, those shown in Table 2.
[Table 2]

[Table 3]

      [0015]
  Other specific examples include those shown in Table 3 below.
[Table 4]

[Table 5]

[Table 6]

[Table 7]

[Table 8]

[Table 9]

[Table 10]

[Table 11]

[Table 12]

[Table 13]

      [0016]
  When creating a database, it is better to select as many toxic substances as possible when adding, but preferably about 20 to 30 toxic substances are selected from the above toxic substances.
  The concentration of the toxic substance used is a concentration that inhibits the growth of the microorganism to be used but does not cause death, and the IC50 value of each toxic substance is usually used. Experiments using living organisms vary in their results. Therefore, when obtaining the IC50 value, three or more experiments are performed and the average value is calculated. Therefore, in order to eliminate experimental errors derived from living organisms, cells were notRawOne possible method is to determine the concentration conditions that affect growth.
  When the microorganism is yeast, the concentration of the toxic substance can also be determined as follows. A dilution series of the toxic substance is prepared, loaded on cells in the initial logarithmic growth phase (OD660 = 0.2-0.5), and further cultured at 25 ° C. for 4 hours. At this time, cells not loaded with the toxic substance are similarly cultured and used as a control. Two hours later, a part of the culture solution to which each concentration of the toxic substance was added and a part of the culture solution of the control group were taken and inoculated on a YPD agar medium. And control samplesCFUCFU-cont). The remainder of the sample is subjected to measurement for database creation, and only data under the optimal conditions determined afterwards are used as data.
  Immediately before adding the toxic substance, a part of the culture solution is taken and seeded on a YPD agar medium. Similarly, two hours later, a part of the culture solution to which the chemical substance of each concentration was added and a part of the culture solution of the control section were taken and inoculated on a YPD agar medium, and two days later, the CFU was measured. CFU-before, CFU of toxic substance loaded sample to CFU-chem, and control sampleCFUCFU-cont). Since the doubling time of yeast is about 2 hours, CFU-cont is usually about 2 times that of CFU-before. When a different concentration of the toxic substance is loaded, a concentration of CFU that affects cell growth, that is, a concentration of 50% or more of CFU-cont, preferably a concentration close to 75.5% is selected as the toxic substance concentration (cell CFU is 0% at the concentration that kills C, and at the concentration that does not affect growth, the CFU is 100%, which is equivalent to the control.)
      [0017]
  The preparation of a "culture medium" for use in creating a database is well known to those skilled in the art. For example, solutions of various compositions selected as described above are sterilized by autoclaving at 121 ° C. for 20 minutes. Amino acids are individually prepared in the form of a high-concentration aqueous solution and sterilized, and are added as necessary so that the final concentration is as shown in Table 1 above. The toxic substance is dissolved in water or dimethyl sulfoxide, and the final concentration is prepared in a dilution series as described above, and the experiment is carried out.
      [0018]
  “Culture of microorganisms” used for database creation is usually performed at 25 ° C. for 4 hours.
      [0019]
  The `` microorganism '' used for database creation includes naturally occurring wild-type or wild-type strains, such as animal cells derived from humans, mice and other mammals, and established animal cell strains, fish, and lines that have been used in bioassays. Any of insect cells, insect cells, fungal cells such as yeast, and bacterial cells such as Escherichia coli may be used. Here, a wild-type strain is a naturally occurring non-recombinant microorganism. The wild-type strain is a microorganism in which the gene of interest has not been modified. For example, W303 has been recombined for auxotrophy and the like, but no further recombination has been performed. Established strains include those that can be subcultured with animal cells, for example, those that have not been recombined, such as cancer cells.
  When naturally occurring cells or established cell lines are used without transformation, the "response exhibited by the microorganism" used to create the database is the mRNA levels of the genes that fluctuate in the presence of toxic substances. mRNA levels were determined by Northern blotting (Nobukun Ogata, Hiroshi Nojima: Genetic Engineering Keyword Book Revised 2nd Edition, Yodosha, 2000, pp299-301), reverse transcription PCR (RT-PCR) (Yusaku Nakabeppu, etc.) It can be measured by Cell Engineering Separate Volume Tips Series Revised PCR Tips, Shujunsha, 1999, pp25-43).
  Here, in the case of yeast cells, "genes that fluctuate in the presence of toxic substances" are disclosed in public databases (for example, MIPS: Munich Information Center for Protein Sequence in Germany, SGD: Saccharomyces Genome Database in the United States). The yeast genes can be selected appropriately from the available yeast genes, and these can be known via the Internet. Preferred genes are mitochondrial proteins, gene repair proteins, energy proteins, transport facilitating proteins, stress proteins, metabolic proteins, detoxicating proteins and genes of unknown function. For example, YKL071W, YCR102C, YOR382W, YLL057C, YLR303W, and the like.
      [0020]
  The “microorganism” used for preparing the database can also be a recombinant of the above-described naturally occurring cell, that is, a transformant. The transformant used in the present invention includes a polynucleotide construct obtained by operably linking a polynucleotide encoding a marker protein to the polynucleotide sequence of the promoter of the “gene that fluctuates in the presence of a toxic substance”. It can be prepared by introducing a vector into the naturally occurring cells described above. Methods for operably linking a polynucleotide encoding a protein to a promoter are well known (for example, RW Old, SB Primrose Principles of Genetic Manipulation, 5th edition, Baifukan, pp138-165, pp.234-263, 2000 reference).
      [0021]
  In connection with the sequence of the promoter, for example, when yeast is used, the entire DNA sequence of the yeast has been disclosed. Therefore, by using a public database (SCPD: The Promoter Database of Saccharomyces cerevisiae), the yeast gene sequence including the promoter can be used. Can be obtained.
  Examples of marker proteins include GFP (Green Fluorescence Protein) (Heim, R., Cubitt, AB and Tsien, RY (1995) Nature 373, 663-664; Heim, R., Prasher DC. And Tsien, RY (1994) Proc. Natl. Acad. Sci., 91, 12501-12504; Warg, S. and Hazerigg, T. (1994) Nature 639, 400-403; Youvan, DC and Michel-Beyerle, ME (1996) Nature Biotechnology 14 1219. -1220; Chalfie, M., Tu, Y., Euskirchen, G., Ward, WW and Prasher, DC (1994) Science 263, 802-805), β-galactosidase (Canestro C, Albalat R, Escriva H, Gonzalez) -Duarte R. Endogenous beta-galactosidase activity in amphioxus: a useful histochemical marker for the digestive system.Dev Genes Evol 2001 Mar 211 (3): 154-6), luciferase (Arch Toxicol 2002 Jun; 76 (5-6): 257-61, Estrogenic activity of UV filters determined by an in vitro reporter gene assay and an in vivo transgenic zebrafish assay.Schreurs R, Lanser P, Seinen W, Van Der Burg B.), and acetyltransferer Z (J Recept Signal Transduct Res 2001 Feb; 21 (1): 71-84, A simplified method for large scale quantification of ranscriptional activity and its use in studies of steroids and steroid receptors. Zhang S, Lu J, Iyama K, Lo SC, Danielsen M.).
      [0022]
  For preparing a vector, for example, in the case of a yeast cell, a plasmid capable of replicating in yeast is selected, a polynucleotide encoding a marker protein is inserted into the plasmid, and then a polynucleotide containing a promoter sequence is inserted into an upstream portion of the marker gene. It can be done by doing.
  Transformants can be prepared by well-known techniques. For example, a method for transforming yeast cells is described in Kaiser C, Michaelis S, Mitchell A: A Cold Spring Harbor Laboratory Course Manual 1994 edition (Cold Spring Harbor Laboratory Press) pp. 133-134, 1994.
  When a transformant is used, the “response indicated by the microorganism” used for preparing the database of the present invention is the expression level of the marker protein. The expression level of the marker protein can be obtained by pulverizing the transformant to obtain a protein extract and measuring the amount of the marker protein in the liquid. For example, when the marker protein is GFP, the amount of fluorescence of the protein extract is measured by a fluorescence spectrophotometer. In addition, the expression level of the marker protein can be directly measured without crushing the transformant by image processing using a fluorescence microscope or laser microscope, measurement using flow cytometry, measurement using evanescent light, and detection using a colorimeter. Cell fluorescence (GFP)AlsoIn some cases, color development (β-gal) can be measured.
      [0023]
  Preferred transformants are yeast that have been transformed with a vector comprising a polynucleotide operably linked to a promoter encoding a yeast gene to a polynucleotide encoding a marker protein, or a chromosomal ORF that is a polynucleotide encoding a marker protein. Yeast that has been transformed to replace, more preferably, the yeast gene is a mitochondrial protein, a gene repair system protein, an energy system protein, a transport promoting protein, a stress protein, a metabolic system protein, a detoxifying protein and It is selected from genes whose functions are unknown.
      [0024]
  "Responses shown by microorganisms" used for database creationDifferenceThe indexing of "" will be described below by taking as an example the case where GFP is used as a marker protein.
  After contact with the toxic substance, the expression level of the marker protein is measured as described above. The yeast cells are washed with physiological saline, then fixed with physiological saline containing 5% formalin, and the fluorescence of 10,000 yeast cells per experiment is determined using flow cytometry (eg, EPICS XL: Beckman Coulter). And the average value of the fluorescence intensity of these cells is obtained. The ratio of the fluorescence intensity of the control group to the average value is determined. For example, if the ratio is 1.5 or less, "-"; if the ratio is 1.5-2.0, "+"; And so on.
      [0025]
  As described above, the obtained data can be put together in a table or an electronic information format and used as a database.
      [0026]
  The present invention relates to a method for using the database of the present invention prepared as described above, wherein the amino acid composition and / or the sugar composition in a culture medium for growing microorganisms are provided.Due to the differenceMicroorganismsFor chemical substancesIndicating responseDifferenceIs used as an indexChemistrySubstancedetectionAlso provided is a method of using the database to match against an indexed response.
      [0027]
2)ChemistryMaterialdetectionMethod
  The present invention further provides an amino acid composition and / or a sugar composition in a culture medium for growing a microorganism.Due to the differenceMicroorganismsFor chemical substancesIndicating responseDifferenceIs used as an index,ChemistrySubstancedetectionOn how to do. Specifically, the chemical substances in the test sampledetectionUsing a microorganism to do
(1) culturing a microorganism in a plurality of culture media having the same amino acid composition and sugar composition in the culture medium used to create the database of the present invention in the presence of the test sample;
(2) Responses of microorganisms in individual culture mediaDifferenceAre indexed,
(3) Indexed responseDifferenceAgainst the index of the database,
Thereby, a chemical substance in the test sample showing the same index as the index in the database is obtained.detectionOn how to do it.
      [0028]
  The present invention also provides a method for detecting chemical substances in a test sample.detectionUsing a microorganism to do
(1) For each culture medium having various amino acid compositions and various sugar compositionsspecificAdd toxic substancesWasPrepare multiple culture media,
(2)IndividualIn the obtained culture medium, in the presence of the test sample, cultured microorganisms for a certain period,
(3) Responses of microorganisms in individual culture mediaDifferenceAre indexed,
(4) Indexed responseDifferenceThe chemical substance in the test sample isdetectionTo a method characterized by:
      [0029]
  Of the present inventionChemistryMaterialdetectionIn the method, "Amino acid composition and preparation of culture medium", "Toxic substance", "Microorganism and its culture", "Response exhibited by microorganism"DifferenceAnd indexing thereof ”is the same as the description of the database creation according to the present invention.
      [0030]
  As described above, the present invention provides a composition of a culture medium used for growing a microorganism.Due to the differenceIt is based on the finding that the response of microorganisms to chemicals is different. Of the present inventionChemistrymaterialdetectionThe method is detectable by altering the composition of the culture medium.ChemistryIt is possible to increase the types of substances.
      [0031]
    【Example】
  Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.
      [0032]
Reference Example 1
Reporter Gene Assay
  Yeast geneYBR072WPrimers for amplifying by PCR the polynucleotide containing the promoter sequence of the above (disclosed in the Promoter Database of Saccharomyces cerevisiae: SCPD). Primers are Oligo4.0-S, Sequencher software for primer design_IDesigned using a Macintosh version, the base sequence of the upper primer is
          GCAGTCAACGAGGAGCGAATCA (SEQ ID NO:1)
And the base sequence of the lower primer is
          GGGGTACCCCGTTAATTTGTTTAGTTTGTTTG (SEQ ID NO:2)
And PCR used a yeast chromosome (Saccharomyces cerevisiae S288C, Cat. 40802, Research Genetics, Inc.) as a template, and used a commercially available kit (KOD DNA Polymerase; code KOD-101, Toyobo) as a reagent.
  The vector used is pYES2 (pYES2, Cat no: V825-20, Invitrogen Corporation, USA), a YEp-type shuttle vector that is replicated in both Escherichia coli and yeast (RW Old, SB Primrose Principles of Genetic Manipulation, Fifth Edition) , Baifukan, pp.234-263, 2000)). The polynucleotide encoding the marker protein GFP is a GFP portion (SEQ ID NO: 54) of the vector pQBI63 (Cat no. 54-0082, Wako Pure Chemical Industries, Ltd.).3) Was used. First, a vector was prepared in which a GFP polynucleotide was inserted into a multiple cloning site of pYES2. Next, the portion of the GAL1 promoter of pYES2 was replaced with a polynucleotide containing the promoter sequence of YBR072W, a yeast gene, to obtain a desired plasmid vector. Insertion of a polynucleotide containing GFP and a promoter sequence was performed by selecting an appropriate restriction enzyme.
      [0033]
  Next, yeast Saccharomyces cerevisiae W303 (American Type Culture Collection ATCC 2001239) was transformed with this plasmid vector. The procedure of the transformation is shown below.
1) The yeast cells Saccharomyces cerevisiae W303 are cultured with shaking in 200 ml of YPD medium until the OD660 becomes 0.5.
2) Collect and suspend in 5 ml of TE-buffer.
3) Add 250 μL of 2.5 M lithium acetate.
4) Dispense 300 μl each, add 10 μl of the above plasmid vector, and incubate at 30 ° C. for 30 minutes.
5) Add 700 μl of 50% PEG4000 and culture with shaking at 30 ° C. for 60 minutes.
6) After heat shock (42 ° C, 5 minutes), cool rapidly.
7) Wash twice with 1 M sorbitol.
8) Seed on an agar plate prepared with a minimal nutrient medium (SD medium supplemented with necessary amino acids (adenine, histidine, tryptophan)).
      [0034]
  Transformation was confirmed using a selection medium (SD medium (Yeast nitrogen base without amino acids (Difco 0919-15) + glucose + amino acids (adenine, histidine, tryptophan)). Colonies that grew on agar plates of the selection medium were further The nutritional requirements of amino acids were confirmed.
  It was confirmed that the obtained transformant stably retained the plasmid on a YPD medium (1% yeast extract, 2% polypeptone, 2% glucose) for up to 48 hours.
      [0035]
Example 1
Chemical loading experiment
  The transformant prepared in Reference Example 1 is brought into contact with each compound having the concentration shown in Table 4 below under the following culture conditions.
[Table 14]
                        Table 4
Effect of chemicals on growth potential of yeast W303 (IC50)
Chemical concentration
(1) 2-aminoanthracene 33.3 mM
(2) Benzo (a) pyrene 0.2 mM
(3) Bisphenol A 0.4 mM
(4) Di (2-ethylhexyl) phthalate 83.3 mM
(5) 2,4-dichlorophenol 0.3 mM
(6) 2,4-dichlorophenoxyacetic acid 0.3 mM
(7) Formaldehyde 0.2 mM
(8) Methylmercury chloride 0.2 μM
(9) 4-nitroquinoline-N-oxide 0.6 μM
(10) p-Nonylphenol 10 μM
(11) Pentachlorophenol 50 μM
(12) Sodium arsenite 0.3 mM
(13) Thiuram 20 μM
(14) Tributyltin chloride 0.4 μM
(15) 2,4,5-trichlorophenol 30 mM
(16) Trp-P-2 (acetate) 0.2 mM
(17) Paraquat 16.7 mM
(18) Cadmium chloride 40 μM
(19) Lindane 6.7 mM
(20) Malathion 22.2 mM
(21) Maneb 0.8 mM
(22) Nickel (II) chloride 3.3 mM
(23) Potassium dichromate (VI) 0.3 mM
(24) Triphenyltin chloride (IV) 10 μM
(25) Phenol 5.6 mM
(26) Thiobencarb 0.7 mM
(27) Hexachlorophene 30 μM
(28) Triclosan 730 μM
(29) Mercury (II) chloride 50 μM
(30) Copper (II) sulfate 3.3 mM
(31) Potassium cyanide 16.7 mM
(32) dimethyl sulfoxide 3.7%

  IC50 was measured in YPD medium and SD medium, and it was confirmed that they were almost equal.
      [0036]
Culture conditions
  Composition of culture medium
  1. SD medium (Yeast nitrogen base without amino acids (Difco 0919-15)) + glucose + amino acids (adenine, histidine, tryptophan);
  2. SD medium + glucose + amino acids (adenine, histidine, tryptophan, uracil)
  3. SD medium + glucose + amino acids (adenine, histidine, tryptophan, leucine)
  4. SD medium + glucose + amino acids (adenine, histidine, tryptophan, methionine)
  5. YPD medium (1% yeast extract, 2% polypeptone, 2% glucose); and
  6. SD medium + galactose + amino acids (adenine, histidine, tryptophan);
      [0037]
  The transformant cryopreserved in 10 ml of SD medium containing the necessary amino acids is scraped off with a platinum loop, shake-cultured at 25 ° C., and allowed to grow to a full-growth state (a state in which the cells grow to a steady state and the number of cells does not increase any more). Add 50ul of the cell suspension which had been fully grown to 10 ml of each culture medium, culture with shaking at 25 ° C, and load the chemical when the OD660 became 0.2 to 0.5. The cells were washed once with physiological saline, then fixed with physiological saline containing 5% formalin, and the fluorescence of 10,000 yeast cells per experiment was determined using flow cytometry (EPICS XL: Beckman Coulter). The average value of the fluorescence intensity of these cells was obtained, and the ratio to the average value of the fluorescence intensity of the control group was determined and indexed.
  Table 5 shows the obtained results. In the table, the fluorescence intensity of the control was 1.5 times or less,-, 1.5 times or more and less than 2.0 times +, and 2.0 times or more ++.
      [0038]
[Table 15]

[Table 16]

      [0039]
  As described above, it has been proved that the growth state of the transformant for each chemical substance varies depending on the composition of the medium to be cultured even when the same transformant is used. The present invention provides the data of Table 5 created as described in the examples as an embodiment of a database.
      [0040]
Example 2
Chemical substance detection in test samples
  A test sample is added to a culture medium having each composition as in Example 1, the transformant is cultured, and the growth state is indexed according to the above-mentioned criteria to obtain an index pattern. As a result, chemical substances showing the same pattern can be specified, and chemical substances that can be present in the test sample are estimated.
  Multiple samples in the test sampleChemistryIf a substance is included, the response exhibited by the microorganism may be additive or synergistic, depending on the combination of types of toxicity. However, in practice, multipleChemistrySince the condition of a substance is very rare, it is considered that the most toxic chemical substance is detected.
  When the concentration of the chemical substance present in the test sample is low, the gene expression is not different from that in the control group, and when the concentration is high, the cells are killed and the gene is not expressed. Therefore, the concentration around IC50 is a suitable concentration for the present invention.
      [0041]
Method for determining a suitable concentration for a test sample
1. How to determine the concentration in advance
  When the test sample has a low concentration, it is concentrated to form a dilution series, and when the test sample has a high concentration, a dilution series is prepared, and the experiment is performed.
  A sample of the dilution series is made and full-grown cells are loaded into a solution diluted 200-fold in minimal nutrient medium. Measure OD660 every 48 hours for up to 48 hours. At this time,RawAt concentrations that affect growth but do not kill cells.RawThe concentration that inhibits growth by about 50% is selected as the IC50.
      [0042]
2.ChemistrymaterialdetectionHow to determine the concentration at the same time
  Further, by measuring the CFU as described below, a suitable concentration can be selected.
  Immediately before adding a chemical substance, a part of the culture solution is taken and inoculated on a YPD agar medium. Similarly, two hours later, a part of the culture solution to which the chemical substance of each concentration was added and a part of the culture solution in the control section were inoculated on a YPD agar medium, and two days later, the CFU was measured (the CFU of the sample immediately before the chemical substance loading was measured). CFU-before, CFU of chemical loaded sample to CFU-chem, and control sampleCFUCFU-cont). Since the doubling time of yeast is about 2 hours, CFU-cont is usually about twice as large as CFU-before. When a different concentration of the chemical is loaded, a CFU that affects cell growth, that is, a concentration of 50% or more and 95% or less of CFU-cont, preferably a concentration close to 75.5% is selected as the concentration of the chemical. (The concentration of CFU is 0% at the concentration at which cells are killed, and the concentration of CFU at the concentration that does not affect growth is 100%, which is the same as in the control group.) The cells to which the chemical substance of each concentration was added were washed once with a saline solution 4 hours after the addition of the chemical substance, then fixed in a physiological saline solution containing 5% formalin, and refrigerated. Data is collected after selecting the concentration. Instead of measuring CFU, a method of counting living cells using a staining method that stains only living cells can be considered.
      [0043]
    [Sequence list]
                               SEQUENCE LISTING
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<212> DNA
<213> Artificial Sequence

<400>Two
ggggtacccc gttaatttgt ttagtttgtt tg 32

<210>Three
<211> 720
<212> DNA
<213> Aequorea victoria
<400>Three
atggctagca aaggagaaga actcttcact ggagttgtcc caattcttgt tgaattagat 60
ggtgatgtta acggccacaa gttctctgtc agtggagagg gtgaaggtga tgcaacatac 120
ggaaaactta ccctgaagtt catctgcact actggcaaac tgcctgttcc atggccaaca 180
ctagtcacta ctctgtgcta tggtgttcaa tgcttttcaa gatacccgga tcatatgaaa 240
cggcatgact ttttcaagag tgccatgccc gaaggttatg tacaggaaag gaccatcttc 300
ttcaaagatg acggcaacta caagacacgt gctgaagtca agtttgaagg tgataccctt 360
gttaatagaa tcgagttaaa aggtattgac ttcaaggaag atggcaacat tctgggacac 420
aaattggaat acaactataa ctcacacaat gtatacatca tggcagacaa acaaaagaat 480
ggaatcaaag tgaacttcaa gacccgccac aacattgaag atggaagcgt tcaactagca 540
gaccattatc aacaaaatac tccaattggc gatggccctg tccttttacc agacaaccat 600
tacctgtcca cacaatctgc cctttcgaaa gatcccaacg aaaagagaga ccacatggtc 660
cttcttgagt ttgtaacagc tgctgggatt acacatggca tggatgaact gtacaactga 720