한국양식학회지 (Journal of Aquaculture)
- 제8권3호
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- Pages.209-220
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- 1995
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- 1226-0193(pISSN)
미꾸라지로부터의 복제원점 클로닝 및 그 특성에 관한 연구
Cloning and Characterization of Replication Origins from Misgurnus mizolepis
- Lim Hak-Seob (Department of Biotechnology and Bioengineering, National Fisheries University of Pusan) ;
- Kim Moo-Sang (Department of Biotechnology and Bioengineering, National Fisheries University of Pusan) ;
- Lee Hyung-Ho (Department of Biotechnology and Bioengineering, National Fisheries University of Pusan)
- 발행 : 1995.08.01
초록
미꾸라지의 간으로부터 핵을 분리하여, 저농도 염추출 및 제한효소 처리로 핵기질(nuclear matrix)을 분리하였다. 분리된 핵기질을 Proteinase K로 분해한 후, phenol-chloroform 추출로 크기가 약 0.3kb-15kb의 분포를 나타내는 핵기질 부착 DNA (nuclear matrix attachment regions : MARs)를 얻었다. 효모 URA 3 유전자를 가진 2.13 kb Eco47 III 단편을 제한효소 Ssp I 으로 절단된 pUC19 플라즈미드 벡타에 결합시켜, ARS (autonomously replication sequence) 클로닝을 위한 pURY19 플라즈미드 벡타를 만들었다. 이 pURY19 벡타는 Saccharomyces cerevisiae내에서 독립적으로 복제할 수 없기 때문에, 물고기의 효율적인 발현 벡타 개발을 위해, 이 system을 이용하여, S. cerevisiae내에서 독립적으로 복제 가능한 미꾸라지의 ARS를 클로닝하고자 하였다. 분리 된 MARs를 pURY19 벡타에 결합시 킨 다음, E. coli
The nuclear matrix was isolated from Misgumus mizolepis liver nuclei by low salt extraction and restriction enzyme treatment. The structure was digested with proteinase K. After centrifugation, matrix attachment regions (MARs) were obtained by RNase treatment and phenol-chloroform extraction. The result leads to the appearance of smeared bands in the range of about 0.3-15 kb. pURY19 vector was constructed by inserting 2.13 kb Eco47 III fragment of the yeast uracil 3 gene into the unique Ssp I site of pUC19 plasmid vector as a selection marker. This vector is unable to be maintained in Sacrharomyces cerevisiae by itself since it cannot replicate as an extrachromosomal element. Using this system, we attempted cloning the ARS (autonomously replicating sequence) from M. mizelepis to develop an efficient expression vector for the transgenic fish.
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