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엄재영,박충웅,이강민 全北大學校 基礎科學硏究所 1994 基礎科學 Vol.17 No.-
DNA를 인식하여 절단하는 제한효소의 발견은 실험실에서 유전자를 연구, 조작할 수 있게되어 분자 생물학 연구에 큰 발전을 가져왔다. 제한효소의 인식자리는 반응용액의 산도, 이온세기, 소수성, 유기용매, 효소의 양에 따라서 달라질 수 있다. 본 연구는 유전공학에서 가장 많이 이용되고 있으며 그의 3차 구조가 밝혀진 EcoRI 제한효소가 유기용매에 의한 특이성 변화를 연구하였다. 이 효소의 특이성은 에탄올, ethyleneglycol, DMSO와 같은 유기용매에 의하여 변화되며, 이 변화는 유기용매의 소수성(LogP)값과 밀접히 관계있다. EcoRI의 용기용매에 의한 특이성변화는 LogP값이 -2.0∼0 사이에서 일어난다. Acetone, 2-methy-propanol같은 그 효소를 쉽게 비활성화시키는 유기용매는 특이성을 변화에 영향을 주지 못한다. 이러한 특이성변화는 무질서하게 일어나지 않고 순서적으로 일어난다. 10% DMSO에 서 EcoRI을 이용하여 pGEM3를 절단할 때 다음 절단자리는 TAATTC, GAGTTC순서로 절단된다. 이와 같은 제한효소의 반응조건을 바꾸면 고유의 절단자리가 아닌 다른자리를 절단할 수 있으며 이러한 기술은 유전공학에 이용될 수 있다. In molecular biology, type-II restriction endonuclease, which specifically cleave DNA at a limited number of sites, have been exploited as a means of characterizing DNA fragments. DNA mapping and of modifying DNA for genetic engineering. Recently, many type-II restriction endonucleases have been found to decrease their substrate specificity under modified conditions such as extreme pH, low ionic strength, high enzyme concentration, substitution of metallic cofactors, or addition of organic solvents. This study used restriction endonuclease EcoRI which are used most frequently in genetic engineering. We investigated their specificity change in buffer condition including various organic solvents. The specificity of cleavage of EcoRI is altered in the presence of hydrophobic reagents, such as ethanol, ethyleneglycol and DMSO. The enzyme recognition site was not changed randomly but by preferential order by increasing the concentration of organic solvent. When EcoRI reacted with substrate pGEM3 vector which have on canonical recognition site (GAATTC). EcoRI cleaved noncanonical TAATTC and GAGTTC subsequently in more than 10% DMSO solution. These changes of specificities depended on the hydrophobicity of organic solvent (LogP : partition coefficient). As a results, the recognition sequence site was changed in the presence of organic solvents whose LogP are -2.0∼0. The specificities were not easily changed in enzyme inactivating organic solvent such as acetone, 2-methyl-2-propanol, 2-methyl-1-propanol. These results might show that restriction enzyme could be used to cleave at unusual site by changing the reaction condition
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엄재영,박충웅,이강민 ( Jaeyoung Um,Chungung Park,Kagmin Lee ) 생화학분자생물학회 1994 BMB Reports Vol.27 No.5
In molecular biology, type-II restriction endonuclease, which specifically cleave DNA at a limited number of sites, have been exploited as a means of characterizing DNA fragments, DNA mapping and of modifying DNA for genetic engineering. Recently, many type-II restriction endonucleases have been found to decrease their substrate specificity under modified conditions such as extreme pH, low ionic strength, high enzyme concentration, subsfitution of metallic cofactors, or addition of organic solvents. This study used restriction endonuclease EcoRI which are used most frequently in genetic engineering. We investigated their specificity change in buffer condition including various organic solvents. The specificity of cleavage of EcoRl is altered in the presence of hydrophobic reagents, such as ethanol, ethyleneglycol and DMSO. The enzyme recognition site was not changed randomly but by preferential order by increasing the concentration of organic solvent. When EcoRl reacted with substrate pGEM3 vector which have one canonical recognition site (GAATTC), EcoRl cleaved noncanonical TAATTC and GAGTTC subsequently in more than 10% DMSO solution. These changes of specificities depended on the hydrophobicity of organic solvent (Loge : partition coefficient). As a results, the recognition sequence site was changed in the presence of organic solvents whose Loge are -2.0∼0. The specificities were not easily changed in enzyme inactivating organic solvent such as acetone, 2-methyl-2-propanol, 2-methyl-l-propanol. These results might show that restriction enzyme could be used to cleave at unusual site by changing the reaction condition.
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