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Dahmus, Michael E.,Kim, Woo-Yeon 中央大學校 遺傳工學硏究所 1995 遺傳工學硏究論集 Vol.8 No.1
HeLa 핵인자와 정제된 송아지흉선 RNA polymerase Ⅱ에 의한 재조합 전사체계의 확립을 위해 자체의 RNA polymerase Ⅱ를 HeLa S-100에서 제거하였다. HeLa S-100을 heparin-Sepharose CL-4B 칼럼에 loading한 후 0.6M KCl로 용리한 분획을 다시 DEAE-5PW 칼럼에서 분획하여 HeLa 세포 자체 RNA polymerase Ⅱ의 90%를 제거할 수 있었다. 여기에 정제된 송아지흉선 RNA polymerase Ⅱ를 첨가하면 in vitro 전사가 다시 가능하였으며 전사촉진인자를 첨가하여 전사된 RNA양이 여러 배수 증가함을 알 수 있었다. In n effort to reconstitute in vitro transcription system using HeLa nuclear factors and purified calf thymus RNA polymerase Ⅱ, endogenous RNA polymerase Ⅱ was removed from HeLa S-100. HeLa S-100 was fractionated on heparin-Sepharose CL-4B. Transcriptionally active fraction(H0.6), which was eluted with 0.6M KCl, was loaded onto DEAE-5PW and step-eluted with KCl. More than 90% of the endogenous RNA polymerase Ⅱ in H0.6 fraction could be removed from the fractions containing active transcription factors. Exogenous RNA polymerase Ⅱ(purified calf thymus RNA polymerase Ⅱ) could reconstitute in vitro transcription reaction with the fractions containing active transcription factors. Stimulatory factor, which is one of the transcription factor, was also shown to enhance the transcription several fold.
Effect of the Chelating Agents on the Phosphorylated Form of RNA Polymerase Ⅱ during Purification
Dahmus, Michael E.,Kim, Woo-Yeon 中央大學校 遺傳工學硏究所 1994 遺傳工學硏究論集 Vol.7 No.1
인산화된 calf thymus RNA polymeraseⅡ는 높은 농도의 chelating agent를 사용하여 정제될 수 있다. 본 연구의 목적은 높은 농도의 chelating agent가 calf thymus DNA를 이용한 비선택적 역가에 미치는 영향을 조사함에 있다. Calf thymus 핵 추출물의 4℃와 37℃에서의 변화를 protein blotting으로 조사한 결과, RNA polymerase Ⅱ0는 단백질 분해효소 저해제들에 의해 약간 안정화되었으며 20 mM EDTA와 10 mM EGTA 존재하에서는 4℃에서 43시간까지 변화하지 않았다. 높은 농도의 chelating agent 존재하에 Phenyl-Superose와 Mono Q에서 분리정제된 RNA polymerase ⅡO의 Mg²+과 Mn²+의 최적농도를 조사한 결과 0.1 mM EDTA 존재하에 정제된 RNA polymerase Ⅱ와 큰 차이가 없었다. The phosphorylated form of RNA polymerase Ⅱ could be purified from calf thymus using the buffer containing high concentrations of chelating agents. The objective of this study was to investigate the effect of high concentrations of chelating agents on the enzyme activity in nonselective assay using calf thymus DNA as DNA template. Calf thymus nuclear extracts were aged at either 4℃ of 37℃ and the stability of RNA polymerase ⅡO determined by protein blotting. RNA polymerase ⅡO was partially stabilized by the inclusion of protease inhibitors and stable for 43 hr at 4℃ in the presence of 20 mM EDTA and 10 mM EGTA. RNA polymerase ⅡO separated on Phenyl-Superose and Mono Q in the presence of buffer containing 20 mM EDTA and 10 mM EGTA dose not change appreciably the optimal concentration range for Mg²+ and Mn²+ compared to the RNA polymerase Ⅱ purified in the presence of 0.1 mM EDTA.