• Bulletin of the Korean Chemical Society
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• 제27권10호
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• pp.1618-1622
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• 2006

Bacteriophage T7 gp4A' is a ring-shaped hexameric DNA helicase that catalyzes duplex DNA unwinding using dTTP hydrolysis as an energy source. To investigate the effect of single-stranded DNA (ssDNA) on the kinetic pathway of dTTP hydrolysis by the T7 DNA helicase complexed with ssDNA, we have first determined optimal concentration of long circular M13 single-stranded DNA and pre-incubation time in the absence of $Mg^{2+}$ which is necessary for the helicase-ssDNA complex formation. Steady state dTTP hydrolysis in the absence of $Mg^{2+}$ by the helicase-ssDNA complex provided $k_{cat}$ of $8.5\;{\times}\;10^{-3}\;sec^{-1}$. Pre-steady state kinetics of the dTTP hydrolysis by the pre-assembled hexameric helicase was monitored by using the rapid chemical quench-flow technique both in the presence and absence of M13 ssDNA. Pre-steady state dTTP hydrolysis showed distinct burst kinetics in both cases, indicating that product release step is slower than dTTP hydrolysis step. Pre-steady state burst rates were similar both in the presence and absence of ssDNA, while steady state dTTP hydrolysis rate in the presence of ssDNA was much faster than in the absence of ssDNA. These results suggest that single-stranded DNA stimulates dTTP hydrolysis reaction by T7 helicase by enhancing the rate of product release step.

• 대한의생명과학회지
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• 제10권2호
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• pp.65-73
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• 2004

I report that single-stranded antisense as a part of large circular (LC-) genomic DNA of recombinant M13 phage exhibits enhanced stability, sequence specific antisense activity, and no need for target site search. A cDNA fragment (708 bp) of rat TNF-$\alpha$ was inserted into a phagemid vector, and TNF-$\alpha$ antisense molecules (TNF$\alpha$-LCAS) were produced as single-stranded circular DNA. When introduced into a rat monocyte/macrophage cell line, WRT7/P2, TNF$\alpha$-LCAS was able to ablate LPS-induced TNF-$\alpha$ mRNA to completion. The antisense effect of TNF$\alpha$-LCAS was shown to be sequence-specific because expressions of three control genes ($\beta$-actin, GAPDH and IL-1$\beta$) were not significantly altered by the antisense treatment. Further, TNF$\alpha$-LCAS was found to be highly efficacious as only 0.1 $\mu$g (0.24 nM) of TNF$\alpha$-LCAS was sufficient to block TNF-$\alpha$ expression in 1$\times10^5$ WRT7/P2 cells. I have also observed specific antisense activity in reduction of NF-$\kappa$B gene expression. The results suggest that an antisense sequence as a part of single-stranded circular genomic DNA has a specific antisense activity.

• Bulletin of the Korean Chemical Society
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• 제30권8호
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• pp.1724-1728
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• 2009

Severe acute respiratory syndrome coronavirus (SARS-CoV) helicase separates the double-stranded nucleic acids using the energy from ATP hydrolysis. We have measured ATPase activity of SARS-CoV helicase in the presence of various types of nucleic acids. Steady state ATPase analysis showed that poly(U) has two-times higher turnover number than poly(C) with lower Michaelis constant. When M13 single-stranded DNA is used as substrate, the Michaelis constant was about twenty-times lower than poly(U), whereas turnover numbers were similar. However, stimulation of ATPase activity was not observed in the presence of double-stranded DNA. pH dependent profiles of ATP hydrolysis with the helicase showed that the optimal ATPase activities were in a range of pH 6.2 ~ 6.6. In addition, ATP hydrolysis activity assays performed in the presence of various divalent cations exhibited that $Mg^{2+}$ stimulated the ATPase activity with the highest rate and $Mn^{2+}$ with about 40% rate as compared to the $Mg^{2+}$.

• BMB Reports
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• 제38권6호
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• pp.690-694
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• 2005

Transcription termination of the human mitochondrial genome requires specific binding to termination factor mTERF. In this study, mTERF was produced in E. coli and purified by two-step chromatography. mTERF-binding DNA sequences were isolated from a pool of randomized sequences by the repeated selection of bound sequences by gel-mobility shift assay and polymerase chain reaction. Sequencing and comparison of the 23 isolated clones revealed a 16-bp consensus sequence of 5'-GTG$\b{TGGC}$AGANCCNGG-3' in the light-strand (underlined residues were absolutely conserved), which nicely matched the genomic 13-bp terminator sequence 5'-$\b{TGGC}$AGAGCCCGG-3'. Moreover, mTERF binding assays of heteroduplex and single-stranded DNAs showed mTERF recognized the light strand in preference to the heavy strand. The preferential binding of mTERF with the light-strand may explain its distinct orientation-dependent termination activity.

• 생명과학회지
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• 제19권3호
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• pp.305-310
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• 2009

플라스미드 pGKV21에는 229-nt single-strand DNA initiation (ssi) signal이 존재한다. Asymmetric PCR 기법으로 합성된 229-nt ssDNA 단편을 이용하여 실제로 RNA polymerase에 의한 priming ability와 protein interaction을 확인하였다. in vitro primer RNA 합성 실험 결과, 229-nt ssDNA 단편은 filamentous M13 phage의 주형 DNA에서와 비슷한 효율로 시발체 RNA를 합성하였으며, 이 반응은 strand-specific하게 이루어졌다. DNase I footprinting과 gel retardation 실험 결과, RNA polymerase와 SSB 단백질은 229-nt ssDNA 단편에 stable interaction을 하며, 시발체 RNA를 합성하였다. 또한, in vivo 조건 하에서 RNA polymerase의 저해제인 rifampicin을 처리하여 세포내에 ssDNA 중간체가 집적되는 정도를 비교하여 본 결과, 플라스미드 pGKV21은 rifampicin-sensitive RNA polymerase가 상보가닥 합성에 관여 함을 보여 주었다.

• 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.

• 한국가금학회지
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• 제20권4호
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• pp.209-216
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• 1993

본 연구는 다가오는 농축산물 수입개방의 시대를 맞이하여 우리 고유의 맛을 지닌 순수한 재래계의 식별 유지 및 개량하기 위한 과학적인 방법을 제시하고자 실시되었다. 본 연구를 위하여 재래계 및 수입계인 백색 Leghorn종을 line별로 유지하면서 경제형질 (체중, 초산일령, 산란을, 난중)과 표현형 특이성 (체형, 두부, 깃털, 정강이)에 대하여 조사하였다. 또한 최첨단 기법인 유전자 지문 기법을 도입하여 재래계와 수입계의 DNA에 대한 유전자 지문을 실시하였다. 재래계의 경제형질에 있어서 체중은 산란계 품종인 White Leghron과 유사한 특징을 보였고, 초산일령 및 최고 산란기는 수입계보다 다소 늦었으며 평균 산란율 역시 수입계에 비해 다소 떨어졌다. 재래계의 난중은 수입계에 비해 약 10g 정도 가벼웠다. M13mp18 single-stranded DNA를 probe로 사용한 유전자 지문에서, 재래계는 4.4kb와 4.9kb에서 수입계는 10.6kb와 12.4kb에서 품종 고유의 구분이 확실한 다수의 band를 확인 할 수 있었다. 따라서 본 연구에서는 우리 고유의 유전자원인 재래계의 유전자 지문을 수입계의 유전자 지문과 식별할수 있는 것으로 개발하였으며, 향후 이 기술을 이용해서 구체적인 재래계의 유지 및 개량에 대한 방안이 강구되어야 할 것이다.