JP4410563B2 - 酵母のレポーター遺伝子としてのエクオリン - Google Patents
酵母のレポーター遺伝子としてのエクオリン Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Description
14MWのアポエクオリンタンパク質、分子状酸素、および、発光原子団のセレンテラジンを含む(Inouye等、1989年;JohnsonおよびShimomura、1978年;ShimomuraおよびJohnson、1978年)。この複合体に3個のCa2+イオンが結合すると、セレンテラジンはセレンテラミドに酸化され、その際に二酸化炭素と青い光とを放出する(最大放出量は〜466nm)(図7)。
エクオリンは、細胞の機能または胚の発生を妨害しないといわれている(Miller等、1994年)。
Nakajima−Shimada等(Nakajima−Shimada等、1991b)は、アポエクオリンcDNA発現系を用いたSaccharomyces cerevisiaeでの細胞内カルシウムのモニタリングを説明している。ここで、エクオリンは、培地中のストレスまたはグルコース変動に関する細胞内のカルシウムのマーカーとして再度使用された。
GPCR)を活性化しても小胞体からの相当するCa2+の放出はみられない。細胞へα因子が追加されることにより(すなわちGPCRのSte2の刺激)、細胞中で[Ca2+]iが基底レベルの約100nMから数百ナノモル濃度に上昇し、同時にCa2+流入が誘導される。Ca2(+)欠乏媒体で細胞をα因子と共にインキュベートすると、Ca2+流入がかなり減少するが、[Ca2+]iの上昇は検出されない(Iida等、1990年)。このわずかな変異(slide variation)は、我々の実験においては、経路の活性の検出に干渉しない。
フェロモン応答要素は、Saccharomyces cerevisiaeのFUS1遺伝子の基礎的かつフェロモン誘導性の転写に必要であり十分である(Hagen等、1991年)。
(a)交配因子誘導遺伝子のプロモーターの制御下でエクオリンをコードする配列を含む改変酵母細胞、
(b)該改変酵母細胞を化合物と共にインキュベートすること、
(c)エクオリン発現量を測定すること、
を含む。
酵母株として、W303MATa、far::hisG、sst2::URA3FOA,
fus1::HIS3を用いた。
酢酸リチウム法(Ito等、1983年)により酵母株を様々なプラスミドで形質転換した)。
完全長cDNAエクオリン遺伝子をPCRで増幅し、この際、テンプレートとしてキメラのミトコンドリアのエクオリンmtAEQ(この場合、短縮化したN末端はヒトチトクロームCターゲティングシグナルに融合される(Rizzuto等、1992年))を用いた。5’PCRプライマーは、エクオリンの野生型配列の初めの50個のヌクレオチド
と、EcoRI(以下、太字で示す)クローニング部位とを含む。3’プライマーは、クローニング部位を全く含まない。
比較するために、LacZを、エクオリンと同じ方法で同じ発現ベクターにサブクローニングしたが、この際、いかなる内因性遺伝子の5’配列とも融合させなかった(発現レベルを高めるために行われることがある(King等、1990年))。
完全長Escherichia coliのβ−ガラクトシダーゼ遺伝子配列を、フェロモン経路の活性依存性の発現ベクターp78−4PRE(TRP1、2μ)(図4)にクローニングした。
細胞を分配するか、および/または、白色の96−ウェルプレートで100μl量で培養した。
測定時間の30分前に、5μMセレンテラジン(Molecular Probes)溶液10μlを各ウェルに分配し(最終濃度0.5μMになった)、細胞をローディング
した。
次に、プレートを残り30分間を30℃でインキュベートした。
注入システムを備えたルミノメーターでエクオリンを検出した(Luminoskan,Labsystem)。各ウェルにおいて、10mMのCaCl2溶液(Pierce社製のリシス緩衝液Y−PERで希釈した1MのCaCl2)を注入した直後に、15秒間発光シグナルを統合した。
ローディングの中間工程を省くために、分析の最初から培地にセレンテラジンを加えてもよく、その場合、セレンテラジンは濃度0.5μMで加えられる。
細胞を分配するか、および/または、白色の96−ウェルプレートで100μl量で培養した。測定時間に、各ウェルにβ−ガラクトシダーゼ検出ミックス(Gal−screen,Tropix)100μlを加えた。
プレートを、1時間、28℃でインキュベートした。
β−ガラクトシダーゼシグナルをルミノメーターで読み取った;0.5秒間発光シグナ
ルを統合した。
この分析において、2種のレポーター遺伝子の発現は、フェロモン交配経路の活性に依存する(Leberer等、1997年)。FUS1プロモーター領域から増幅された最小プロモーター4PRE(Hagen等、1991年)は、交配シグナルの場合のみ活性化された。このシグナルは、フェロモン受容体Ste2がそのリガンドであるα因子で刺激されることにより惹起される。
p78−4PRE−AEQまたはp78 4PRE−LacZプラスミドのいずれかで形質転換された酵母株の3個のコロニーを、適切な培地(SCグルコース−Trp)で定常期に達するまで培養し、続いて、0;10-7;10-9;10-11Mのα因子(Sigm
a)を含む同培地(エクオリン株に対しては0.5μMセレンテラジンを含む)で希釈し
た。続いて、プレートを、30℃で、700r.p.m.で、3時間、6時間、および、24時間振盪した。
刺激してから3時間後、検出された比率は、すでに1nMまたは100nMのα因子で、β−ガラクトシダーゼよりエクオリンのほうが高かった(それぞれ、エクオリンでは8および24の比率、β−ガラクトシダーゼでは4および7の比率)。刺激してから6時間後、β−ガラクトシダーゼの比率は、同じレベルに留まったが(いずれのα因子濃度においても7の比率であった)、エクオリン検は、より高いレベルが検出された(α因子1nMでは11の比率であり、α因子100nMでは36の比率であった)。刺激してから24時間後、エクオリン数は、β−ガラクトシダーゼより高いレベルを維持した。
すなわち、この実験から、エクオリンが、β−ガラクトシダーゼよりも良好に様々なリガンド濃度でのSte2の刺激を長期間にわたり示すことがわかった。
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Shimomura O and Johnson F H (1978) Peroxidized Coelenterazine, the Active Group in the Photoprotein Aequorin. Proc NatI Acad Sci U S A 75: pp 2611-2615.
Stables J, Mattheakis L C, Chang R and Rees S (2000) Recombinant Aequorin As Reporter of Changes in Intracellular Calcium in Mammalian Cells. Methods Enzymol 327: pp 456-471.
Thomas AP and Delaville F (1991) The Use of Fluorescent Indicators for
measurements of cytosolic-free calcium concentration in cell populations and single cells., in Cellular Calcium: A Practical Approach (McCormack JG and Cobbold PH eds)pp1-54.
Claims (2)
- 交配因子誘導遺伝子のプロモーターの制御下で発現するエクオリンをコードする配列を含む改変酵母細胞であって、
発現が細胞表面タンパク質により仲介され、そして
プロモーターは配列番号2の配列を有するFUS1プロモーターの部分であり、この部分は配列番号5の配列を有するプライマーおよび配列番号6の配列を有するプライマーを用いて野生型酵母ゲノムDNAの該プロモーターを増幅することを含む方法により得ることができる、
改変酵母細胞。 - (a)交配因子誘導遺伝子のプロモーターの制御下でエクオリンをコードする配列を含む改変酵母細胞であって、
プロモーターは配列番号2の配列を有するFUS1プロモーターの部分であり、この部分は配列番号5の配列を有するプライマーおよび配列番号6の配列を有するプライマーを用いて野生型酵母ゲノムDNAの該プロモーターを増幅することを含む方法により得ることができる、
該改変酵母細胞を、
化合物と共にインキュベートすること、および、
(b)エクオリン発現量を測定すること、
を含む、異種細胞表面タンパク質が仲介するエクオリン発現を調節する化合物を同定する方法。
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EP01125709A EP1306385A1 (en) | 2001-10-27 | 2001-10-27 | Aequorin as a reporter gene in yeast |
PCT/EP2002/011632 WO2003037924A1 (en) | 2001-10-27 | 2002-10-17 | Aequorin as a reporter gene in yeast |
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