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Purification and Characterization of a Restriction Endonuclease Zan I from Zymomonas anaerobia
선대규,유욱준,Sun, Dae-Kyu,Yoo, Ook-Joon 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.4
Zymomonas anaerobia (NCI B8227)로부터 type II 제한효소인 Zan I 을 순수 정제하여 그 특성에 대해 연구하였다. 정제된 효소를 0.1% SDS 존재하에서 10% polyacrylamide gel 전기영동을 수행한 결과 그 subunit의 분자량은 약 30,000임이 확인되었다. 제한효소 Zan I은 BstN I 및 EcoR II와 같은 인식부위를 가지고 있었으며 괄호속의 염기배열에서 화살표 후 표시한 위치를 절단하였다(5'-$CC{\uparrow}$ (AT)GG-3'). 이 제한효소의 반응을 위한 최적 온도는 $37^{\circ}C$였으며 dcm methylated $CH_3$ DNA (5'-CC(AT)GG-3')도 정상적으로 절단할 수 있다는 특성도 있음이 발견되었다. We described the purification and characterization of a sequence specific restriction endonuclease, Zan I, found from Zymomonas anaerobia (NCI B8227). The purified enzyme is essentially homogeneous and the subunit molecular weight is $30,000{\pm}1,000$ as judged by 10% polyacrylamide gel electrophoresis containing 0.1% SDS. The recognition sequence and the cleavage site (indicated by the arrow) were determined to be 5'-$CC{\uparrow}$ (AT)GG-3', the same sequence of BstN I and EcoR II. Zan I endonuclease is able to cleave dcm-methylated DNA and is maximally active at the temperature of $37^{\circ}C$.
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Zymomonas anaerobia 로부터 제한효소 Zan I 의 정제 및 촉매적 특성에 관한 연구
선대규,유욱준 ( Dae Kyu Sun,OOk Joon Yoo ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.4
We described the purification and characterization of a sequence specific restriction endonuclease, Zan I, found from Zymomonas anaerobia (NCI B8227). The purified enzyme is essentially homogeneous and the subunit molecular weight is 30,000 ± 1,000 as judged by 10% polyacrylamide gel electrophoresis containing 0.1% SDS. The recognition sequence and the cleavage site (indicated by the arrow) were determined to be 5`-CCl (AT) GG-3`, the same sequence of BstN I and EcoR II. Zan I endonuclease is able to cleave dcm-methylated DNA and is maximally active at the temperature of 37℃.
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Transposon Tn5를 이용한 Slow growing Rhizobium japonicum의 돌연변이 유도
김성훈,이윤,선대규,유익동,Kim, Sung-Hoon,Rhee, Yoon,Sun, Dae-Kyu,Yoo, Ick-Dong 한국미생물학회 1988 미생물학회지 Vol.26 No.4
Slow growing R. japonicum R-l68 균주로부터 spectinomycin 내성 균주를 선발하고 이 Rhizobium내에 Tn-5를 도입시키기 위하여 Tn5가 함유된 E. coli WA 803/pGS9과의 conjugation을 통한 transposon mutagenesis를 실시하였다. 이때 C conjugation을 통한 Tn5 전이 빈도는 $1.0\times 10^{-5}-5.0\times 10^{-7}$ 범위 이였으며, 얻어진 transconjugant들은 spectinomycin (($100{\mu}$g/ml)과 kanamycin ($50{\mu}$g/ml)을 함유한 yeast extract-mannitol 배지에서 8-10일 배양후 colony를 형성하였다. 또한 transconjugant들은 genome상에 Tn-5를 함유하고 있음을 hybridization-을 통하여 확인하였다. 한편 nodule은 형성 하나 질소고정 활성이 없는 돌연변이주 R. japonicum RMa 75 $nod^{+}fix^{-}$ 균주를 선발하였는데 이 균주는 nodule내에 leghemoglobin이 결핍되어 있음이 확인되었다. The spectinomycin resistant strain of slow growing R. japonicum R-168 was selected to be participated in conjugation with E. coli WA803/pGS9. Tn5 was introduced from suicide vector pGS9 into R. japonicum R-168 $spr^{r}$ chromosome at the frequency of $1.0\times 10^{-5}-5.0\times 10^{-7}$ and the transconjugante were selected on the yeast extract-mannitol plate containing kanamycin ($50{\mu}$g/ml) and spectinomycin ($100{\mu}$g/ml) after 8-9 days incubation. All transconjugants we tested were found to contain Tn 5 DNA on their genome, which was confirmed by Southern hybridization experiments. R. japonicum RNa75, which had been selected through plant test, was found to be defective in symbiotic nitrogen fixing ability and the production of leghemoglobin in soybean nodules formed by the inoculation of this mutant. In addition, this mutant strain hardly developed nitrogenase activity asymbiotically in contrast with the wild type strain, indicating that some nitrogen fixing gene might be blocked in this strain and the production of leghemoglobin could be decreased by the interference in nitrogen fixing genes.
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