E. coli ゲノムにおける 2'-デオキシチジン アナログによる 2'-デオキシチジン 代替
Angad P Mehta1, Han Li1, Sean A Reed1
1The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
Journal of the American Chemical Society
|November 3, 2016
まとめ
研究者は,Escherichia coliを63%の2'-デオキシチジン (dC) を5 - 水素メチル-2'-デオキシチジン (5hmC) で置き換えて,エピジェネティック・レギュレーション研究のために20%のゲノム5 - グルコシル-5- 水素メチル-2'-デオキシチジン (5- gmC) を達成しました.
科学分野:
- 合成生物学
- エピジェネティクス
- 微生物遺伝学
背景:
- DNAの改変された核酸は表遺伝的調節と制限システムにとって極めて重要です.
- 以前の研究では,チミジン (T) の75%を,エクストクォーテシングル-ヒドロキシメチル-2エクストクォーテシングル-デオキシウリジン (5hmU) で置き換えた.
研究 の 目的:
- E. coliのピリミジン核酸生物合成経路を設計する
- 2 エクストクォーツシングルデオキシチジン (dC) を 5 水素メチル-2 エクストクォーツシングルデオキシチジン (5hmC) に置き換える研究.
- 5hmCを5-glucosyl-5-hydroxymethyl-2 extquotesingle-deoxycytidine (5-gmC) にさらに改変し,そのゲノムとプラズミドDNAの組み込みを評価する.
主な方法:
- ピリミジンヌクレオチド生物合成経路を設計するために T4 細菌菌遺伝子を利用した.
- グルコースの代謝経路を設計して ヌクレオシドの改変を容易にした
- 分子生物学技術を用いて,ゲノムとプラズミドDNAの両方で核酸置換レベルを定量化した.
主要な成果:
- 大腸菌のゲノムで約63%のdCを5hmCで置き換えた.
- プラズミドDNAで約71%のdCを5hmCで置き換えた.
- ゲノムDNAで20%の5gmCと プラズミドDNAで45%の5gmCを生成しました
結論:
- 改変されたサイトシン誘導体の高レベルの組み込みのためのE. coliの成功エンジニアリングが実証された.
- 5gmC改変DNAを生成するシステムを確立し,その生物学的機能を研究する道を開いた.
- バクテリアのゲノムにおける広範囲の核酸変異の結果を理解するための基礎を提供します.
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