• 미생물학회지
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• 제24권4호
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• pp.406-410
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• 1986

The isolation and charateriation of a type II restriction endonuclease from Xanthomonas citri IFO 3835 were described. This enzyme (Xci I endonuclease) is an isoschizomer of Sal I endonuclease recognizing 5'-GTCGAC-3' and cleaving at the site indicated by the arrow. Unlike Sal I endonuclease, Xci I endonuclease requries a NaCl concentration of 50mM for its maximum activity.

• 미생물학회지
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• 제26권1호
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• pp.1-5
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• 1988

The esolation and characterization of a new type II restriction endounclease, BamI, from Bacellus macerans ATCC 8244 were described. BmaI endonuclease was partially purified by procedures of ammonium sulfate fractionation, DEAE-cellulose and phosphocellulose chromatographies. This enzume recognized one site on pBR322 DNA, two sites on Bluescribe DNA, three sites on $\lambda$DNA and no site on SV 40 DNA. The same cleavage patterns for vareius DNAs as PvuI indicated that BamI is an isoschisomer of PvuI whose recognition sequence is 5'-CGATCG-3'. The optimal pH for the BmaI endonuclease activity was about 7.0 and optimal NaCl concentration was about 100mM. Manganese ion could partially replace magnesium as a cofactor, but calcium could not at all.

• 한국미생물·생명공학회지
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• 제15권5호
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• pp.352-355
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• 1987

새로운 제한효소인 Cth I을 Clostridium thermocellum ATCC 27405로부터 분리하여, 그 효소의 생화학적 특성을 연구하였다. 이 효소는 Bcl I endonuclease의 isoschizomer로서 5'-TGATCA-3'를 인식한다. Cth I endonuclease는 섭씨 60도의 높은 온도에서 잘 작용하며, 10mM까지의 저농도의 NaCl과 MG$^{2+}$ 이온의 존재하에서 pH7.5와 10.5사이에서 최적의 활성을 보여주었다. Cth I endonuclease의 활성은 DNA 기질이 dam methylation 되었을 때 저해를 받았다.

• 대한수의학회지
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• 제33권1호
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• pp.43-51
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• 1993

Mitochondrial DNA(mt DNA) of Mammalian is the circular one which the 16.5K base pairs and show the maternal inheritance. Evolutional speed of nucleotide sequence is very fast. So that polymorphic analysis of mt DNA provide the useful informations to investigate the genetic relations of interspecies. Authors trials were focussed to compare with the polymorphic differences of mitochondrial DNA between Jindo and Japanese mongrel dogs. DNA was extracted from bloods of 21 head of Jindo dogs and 20 head of Japanese dogs and isolated using 10 kinds of restriction endonucleases(Apa I, BamH I, Bgl II, EcoR I, EcoR V, Hinc II. Hind III, Pst I, Sty I, Xba I) and then separated by the agarose gel electrophoresis. After sourthern blotting hybridization was completed using the mtDNA of Japanese mongrel dogs as a probe. Autoradiography was used to compare the polymorphism of mtDNA both dogs. The results obtained were as follows; 1. mt DNA of Jindo dog showed polymorphism resulting cleavage with four kinds of restriction endonuclease, Apa I, EcoR V, Hinc II, Sty I. While in the Japanese mongrel dogs observed the polymorphism in the five kinds of restriction endonuclease supplemented with EcoR I. 2. Compared with both dogs the frequency differences of DNA polymorphism were recognized in the specific restriction endonuclease Apa I. Consequently in the restriction endonuclease Apa I both dogs classified with three types as A, B, C however in the Jindo dogs frequency of C type was 71.5 percent but in Japanese mongrel dogs observed 45 percent in the A type. 3. DNA polymorphism obtained from the use of five kinds of restriction endonuclease were classified with seven types. In Jindo dogs frequency was highest in the type 6 as 71.4 percent but in the Japanese mongrel dogs showed 35 percent in the type 5. 4. Genetic distances calculated by NEI method showed 0.0089 in Jindo dogs and was 0.0094 in the Japanese mongrel dogs.

• 미생물학회지
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• 제25권3호
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• pp.180-183
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• 1987

Type II제한효소 Stu I을 순수정제하고 그 효소적 특성을 연구하였다. 100g(we weight)의 Streptomyces tubercidicus(ATCC 25502)로부터 얻은 crude extranct를 ammonium sulfate fractionation한 후, DEAE-Sephadex(A-50), QAE-Sephadex(A-50) 그리고 Heparin-agarose의 순서로 column chromatography를 수행하여 1,2mg의 비특이성 nuclease가 없는 Stu I 제한효소를 얻었다. 이 시료에 포함되어 있는 다른 오염 단백질은 Sephadex G-100 column으로 gel filtration 하여 제거함으로써, 순수한 Stu I 단백질을 얻을 수 있었다. 정제된 Stu I 제한효소는 10% SDS-polyacrylamide gel electrophoresis 결과 한 개의 band로 나타났으며, 그 분자량은 34,000 $\pm$ 1,000 dalton이었다. 이 효소는 $Mg^{2+}$이온 존재하에 중성의 pH(7.0-8.0)에서 최대의 활성을 나타내었다. NaCl은 이 효소의 활성에는 필요하지 않았다.

• 미생물학회지
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• 제32권4호
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• pp.285-290
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• 1994

자연계에서 새로운 제한효소 생산균을 검색하여 한 균주를 선발하고, 형태학적, 생리학적, 생화학적 특성들을 조사하여 Alcaligenes sp.로 동정하고 제한효소의 특성을 조사하였다. Alcaligenes sp. J-482가 생산하는 제한효소를 AspJI으로 명명하였다. AspJI은 pBR322, Adenovirus 2-DNA, ${lambda}$ DNA 등에 대한 절단양식이 AatII와 같아 AatII의 isoschizomer로 추정 되었으며, 효소활성에 12.5mM 이상의 $MgCl_2$를 필요로 하였으며, NaCl에 의하여 저해되었다. AspJI의 반응 최적 온도는 $37^{circ}C$, 최적 pH는 7.5로 확인 되었으며, 내열성을 조사한 결과 $85^{circ}C$이상에서 15분처리 할 때 안전히 실활되는 것으로 관찰되었다.

• 미생물학회지
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• 제26권2호
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• pp.88-92
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• 1988

The isolation and characterization of a new type II methylase, BmaI methylase, from Bacillus macerans ATCC 8244 were described. BmaI methylase was isolated by procedures of ammonium sulfate fractionation, DEAE-cellulose chromatography and phosphocellulose chromatography. Two types of methylases were present in this strain and only one of the two was a site specific BmaI methylase. The pBR322 DNA methylated by BmaI methylase was not cleaved by BmaI endonuclease, and pBR322 DNA cleaved by BmaI endonuclease was not methylated by BmaI methylase. The optimal pH for the BmaI methylase activity was 7.5, and optimal NaCl concentration was about 50 mM. BmaI methylase could methylate single-stranded M13mp18 DNA.

• 미생물학회지
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• 제32권3호
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• pp.208-214
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• 1994

토양으로부터 분리한 방선균주로부터 새로운 type II 제한효소를 분리하여 그 특성을 연구하였다. 이 균주는 수리분석 결과, Streptoverticillium olivoverticillatum으로 동정되었으며, 정제한 제한효소는 SolI이라 명명하였다. SoII은 BamHI의 isoschizomer로서 여섯 개의 염기서열 5'-G $\downarrow$ GATCC- 3'을 인지하며 두 개의 G 염기 사이에서 절단하여 4 base가 돌출된 5'말단을 생성한다. 그러나 BamHI과는 달리, dam methylation 되어 있는 인식 염기서열에는 작용하지 못하였다. Ammonium sulfate 분획(30-65%)과 heparin-agarose, Affi-gel Blue column chromatography를 거쳐 SolI을 부분 정제하였다. SolI은 활성을 보이기 위하여 0.2mM 이상의 $MgCl_2$를 반드시 필요로 하였으며, 다른 cofactor는 요구하지 않았다. NaCl이 없을 때 가장 높은 활성을 가지며 120 mM 이상의 NaCl이 있으면 활성이 완전히 억제되었다. 이 효소의 반응 최적 온도는 $40^{\circ}C$, 최적 pH는 8.6으로 나타났다. Gel filtration chromatography에서의 용출부피 비교로부터 이 효소의 분자량은 약 43,000Da인 것으로 추정된다.

• 한국자기공명학회논문지
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• 제24권3호
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• pp.70-76
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• 2020

The CRISPR-Cas system provides an adaptive immunity for bacteria and archaea against invading phages or foreign plasmids. In the type II CRISPR-Cas system, a single effector protein Cas9 and a guide RNA form an RNA-guided endonuclease complex that can degrade DNA targets of foreign origin. To avoid the Cas9-mediated destruction, phages evolved anti-CRISPR (Acr) proteins that neutralize the host bacterial immunity by inactivating the CRISPR-Cas system. Here we report the backbone ¹H, ¹⁵N, and ¹³C resonance assignments of AcrIIA5 that inhibits the endonuclease activity of type II-A Streptococcus thermophilus Cas9 and also Streptococcus pyogenesis Cas9 using triple resonance nuclear magnetic resonance spectroscopy. The backbone chemical shifts of AcrIIA5 predict a disordered region at the N-terminus, followed by an αββββαβββ fold.

• 한국자기공명학회논문지
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• 제22권3호
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• pp.71-75
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• 2018

The CRISPR-Cas system is the adaptive immune system in bacteria and archaea against invading phages or foreign plasmids. In the type II CRISPR-Cas system, an endonuclease Cas9 cleaves DNA targets of phages as directed by guide RNA comprising crRNA and tracrRNA. To avoid targeting and destruction by Cas9, phages employ anti-CRISPR (Acr) proteins that act against host bacterial immunity by inactivating the CRISPR-Cas system. Here we report the backbone $^1H$, $^{15}N$, and $^{13}C$ resonance assignments of AcrIIA4 that inhibits endonuclease activity of type II-A Listeria monocytogenes Cas9 and also Streptococcus pyogenesis Cas9 using triple resonance nuclear magnetic resonance spectroscopy. The secondary structures of AcrIIA4 predicted by the backbone chemical shifts show an ${\alpha}{\beta}{\beta}{\beta}{\alpha}{\alpha}$ fold, which is used to determine the solution structure.