• 미생물학회지
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• 제32권2호
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• pp.102-108
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• 1994

ABF1(Autonomously replicating sequence Binding Factor 1)은 효모 genome에서 $RTCRYN_5ACG$의 염기 서열을 가지고 있는 promoter, mating-type silencer, ARS에 결합하는 DNA binding 단백질이다. E. coli 에서 ABF1 유전자를 발현하기 위하여, ABF1 유전자를 pMAL-c2 벡터에 cloning하였다.(pMAHW). pMAHW를 E. coli에 형질전환하여, ABF1 융합단백질을 발현시키고, amylose resin affinity chromatography에 의하여 분리하였다. Factor Xa protease를 이용하여 분리된 융합단백질로부터 maltose binding protein을 잘라낸 후에 gel retardation analysis 방법으로 분리된 ABF1이 ARS1에 결합하는 능력을 지니고 있음을 확인하였다. DNA 결합에 관련된 부위를 찾기 위하여, 비전형적인 zinc finger motif가 위치하는 자리에서 pMAHW의 ABF1 유전자에 His-61을 다른 아미노산으로 치환하였다. DNA binding 부위로 추정되는 ABF1 단백질의 중간지역에 Leu-353, Leu-360를 다른 아미노산으로 치환하였다. Site-specific mutagenesis 를 통해 만들어진 mutant를 gel retardation analysis와 complementation test를 통해서 비전형적인 zinc finger motif이외에 다른 DNA binding motif가 있는 것을 알 수 있었다.

• Toxicological Research
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• 제33권4호
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• pp.265-272
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• 2017

Aryl hydrocarbons such as 3-nitrobenzanthrone (NBA), 4-aminobiphenyl (ABP), acetylaminofluorene (AAF), benzo(a)pyrene (BaP), and 1-nitropyrene (NP) form bulky DNA adducts when absorbed by mammalian cells. These chemicals are metabolically activated to reactive forms in mammalian cells and preferentially get attached covalently to the $N^2$ or C8 positions of guanine or the $N^6$ position of adenine. The proportion of $N^2$ and C8 guanine adducts in DNA differs among chemicals. Although these adducts block DNA replication, cells have a mechanism allowing to continue replication by bypassing these adducts: translesion DNA synthesis (TLS). TLS is performed by translesion DNA polymerases-Pol ${\eta}$, ${\kappa}$, ${\iota}$, and ${\zeta}$ and Rev1-in an error-free or error-prone manner. Regarding the NBA adducts, namely, 2-(2'-deoxyguanosin-$N^2$-yl)-3-aminobenzanthrone (dG-$N^2$-ABA) and N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG-C8-ABA), dG-$N^2$-ABA is produced more often than dG-C8-ABA, whereas dG-C8-ABA blocks DNA replication more strongly than dG-$N^2$-ABA. dG-$N^2$-ABA allows for a less error-prone bypass than dG-C8-ABA does. Pol ${\eta}$ and ${\kappa}$ are stronger contributors to TLS over dG-C8-ABA, and Pol ${\kappa}$ bypasses dG-C8-ABA in an error-prone manner. TLS efficiency and error-proneness are affected by the sequences surrounding the adduct, as demonstrated in our previous study on an ABP adduct, N-(2'-deoxyguanosine-8-yl)-4-aminobiphenyl (dG-C8-ABP). Elucidation of the general mechanisms determining efficiency, error-proneness, and the polymerases involved in TLS over various adducts is the next step in the research on TLS. These TLS studies will clarify the mechanisms underlying aryl hydrocarbon mutagenesis and carcinogenesis in more detail.

• 방사선산업학회지
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• 제6권2호
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• pp.189-197
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• 2012

This study was carried out to develop the improved useful mutants for yield or composition of stevia plants using the gamma ray or chemical mutagens treatments. The seeds of stevia 'Suwon No. 11' were irradiated up to 400 Gy of gamma ray. Chemical mutagens were treated on the seeds of the 'Suwon No. 11' using 0.07% colchicine, 10 mM sodium azide, or 10 mM NMU for various durations. The germination rate, and shoot and root growth of seedling were estimated at 30 days after gamma ray irradiation or chemical mutagen treatment, and the plant height, the number of branches, and leaf length and width were examined at 3 months after mutagenesis treatments. In the case of gamma ray treatments, the germination rate and early-stage growth were decreased as the increase of radiation dose, and the 50% lethal dose was found to be 200 Gy. the plant height was decreased as the increase of radiation dose, while the number of branches per plant and leaf length were increased. Leaf shape was modified to the relatively longer one compared to the control, which was identified more apparently at the treatments of higher than 150 Gy. In the treatment of chemical mutagens, the rate of germination and survival were decreased as the increase of incubation time. The 50% lethal dose for germination rate were identified as the conditions of the 15 hours incubation in 0.07% colchicine, the 4 hrs in 10 mM sodium azide, and the 2 hrs in 10 mM NMU, in the three chemical mutagens treatments. Chemical mutagens had no influence on shoot growth, while root growth was increased, especially as the incubation time was extended. The highest root growth occurred in the NMU treatment at 6 hrs incubation time. The plant height was decreased as the increase of incubation time in the chemical mutagens treatments. Among the chemical mutagens, NMU was the most effective to induce the mutants with long-shaped or the least lobed leaves.

• Molecules and Cells
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• 제41권12호
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• pp.1061-1071
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• 2018

From Xenopus embryo studies, the BMP4/Smad1-targeted gene circuit is a key signaling pathway for specifying the cell fate between the ectoderm and neuro-ectoderm as well as the ventral and dorsal mesoderm. In this context, several BMP4/Smad1 target transcriptional factors have been identified as repressors of the neuro-ectoderm. However, none of these direct target transcription factors in this pathway, including GATA1b, Msx1 and Ventx1.1 have yet been proven as direct repressors of early neuro-ectodermal gene expression. In order to demonstrate that Ventx1.1 is a direct repressor of neuro-ectoderm genes, a genome-wide Xenopus ChIP-Seq of Ventx1.1 was performed. In this study, we demonstrated that Ventx1.1 bound to the Ventx1.1 response cis-acting element 1 and 2 (VRE1 and VRE2) on the promoter for zic3, which is a key early neuro-ectoderm gene, and this Ventx1.1 binding led to repression of zic3 transcription. Site-directed mutagenesis of VRE1 and VRE2 within zic3 promoter completely abolished the repression caused by Ventx1.1. In addition, we found both the positive and negative regulation of zic3 promoter activity by FoxD5b and Xcad2, respectively, and that these occur through the VREs and via modulation of Ventx1.1 levels. Taken together, the results demonstrate that the BMP4/Smad1 target gene, Ventx1.1, is a direct repressor of neuro-ectodermal gene zic3 during early Xenopus embryogenesis.

• 생명과학회지
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• 제29권1호
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• pp.123-128
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• 2019

원핵생물 분류의 기본단위인 종(species)의 동정에 16S rDNA가 사용되지만 한계가 있고 원핵생물의 속(genus)에 대한 연구가 많지 않다. 본 연구에서는 보존 유전자를 확보한 COG database와 대사경로를 확보한 MetaCyc database에 공통적인 원핵생물 중 속이 같고 종이 다른 13개 속 28개의 원핵생물을 대상으로 속 수준에서 연구하였다. 전체 유전자에서 core-genome인 속 보존 유전자의 비율은 최저 27.62%(Nostoc 속)에서 71.76%(Spiribacter 속)의 범위로 평균 46.72%였다. 각 원핵생물에서 core-genome의 비율이 낮으면 특이한 생명현상을 보이거나 서식지가 다양할 수 있을 것이다. 속 수준의 공통 대사경로의 비율은 최저 58.79%(Clostridium 속)에서 최대 96.31%(Mycoplasma 속), 평균 75.86%로 core-genome의 비율보다 높았다. 비교대상을 확장하면 속 특이 보존 유전자와 대사경로는 확인할 수 없었다. 보존 유전자와 대사경로 보유 계통수에서는 대체로 같은 속의 구성원들이 가장 인접하였으며, Bacillus속과 Clostridium 속이 그룹을 형성하였고, 고세균끼리 그룹을 형성하였다. 보존 유전자 보유계통수에서는 Acidobacteria, Cyanobacteria, Proteobacteria 문(phylum)의 Granulicella, Nostoc, Bradyrhizobium의 3개 속이 하나의 그룹을 형성하였다. 본 연구 결과는 (i) 각 계통 단계에서 보존유전자와 대사경로의 확인, (ii) 수평적 유전자 전달 또는 부위 지정 돌연변이를 통한 균주의 개선 등의 분야에 기초자료로 활용될 수 있을 것이다.

• Journal of Microbiology and Biotechnology
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• 제29권3호
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• pp.373-381
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• 2019

Site-directed mutagenesis was employed to generate five different triple point mutations in the double mutant (C295A/I86A) of Thermoanaerobacter ethanolicus alcohol dehydrogenase (TeSADH) by computer-aided modeling with the aim of widening the small alkyl-binding pocket. TeSADH engineering enables the enzyme to accept sterically hindered substrates that could not be accepted by the wild-type enzyme. The underline in the mutations highlights the additional point mutation on the double mutant TeSADH introduced in this work. The catalytic efficiency ($k_{cat}/K_M$) of the ${\underline{M151A}}$/C295A/I86A triple TeSADH mutant for acetophenone increased about 4.8-fold higher than that of the double mutant. A 2.4-fold increase in conversion of 3'-methylacetophenone to (R)-1-(3-methylphenyl)-ethanol with a yield of 87% was obtained by using ${\underline{V115A}}$/C295A/I86A mutant in asymmetric reduction. The ${\underline{A85G}}$/C295A/I86A mutant also produced (R)-1-(3-methylphenyl)-ethanol (1.7-fold) from 3'-methylacetophenone and (R)-1-(3-methoxyphenyl)-ethanol (1.2-fold) from 3'-methoxyacetophenone, with improved yield. In terms of thermal stability, the ${\underline{M151A}}$/C295A/I86A and ${\underline{V115A}}$/C295A/I86A mutants significantly increased ${\Delta}T_{1/2}$ by $+6.8^{\circ}C$ and $+2.4^{\circ}C$, respectively, with thermal deactivation constant ($k_d$) close to the wild-type enzyme. The ${\underline{M151A}}$/C295A/I86A mutant reacts optimally at $70^{\circ}C$ with almost 4 times more residual activity than the wild type. Considering broad substrate tolerance and thermal stability together, it would be promising to produce (R)-1-(3-methylphenyl)-ethanol from 3'-methylacetophenone by ${\underline{V115A}}$/C295A/I86A, and (R)-1-phenylethanol from acetophenone by ${\underline{M151A}}$/C295A/I86A mutant, in large-scale bioreduction processes.

• The Plant Pathology Journal
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• 제35권4호
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• pp.351-361
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• 2019

In our previous study, pyrrolnitrin produced in Pseudomonas chlororaphis G05 plays more critical role in suppression of mycelial growth of some fungal pathogens that cause plant diseases in agriculture. Although some regulators for pyrrolnitrin biosynthesis were identified, the pyrrolnitrin regulation pathway was not fully constructed. During our screening novel regulator candidates, we obtained a white conjugant G05W02 while transposon mutagenesis was carried out between a fusion mutant $G05{\Delta}phz{\Delta}prn::lacZ$ and E. coli S17-1 (pUT/mini-Tn5Kan). By cloning and sequencing of the transposon-flanking DNA fragment, we found that a vfr gene in the conjugant G05W02 was disrupted with mini-Tn5Kan. In one other previous study on P. fluorescens, however, it was reported that the deletion of the vfr caused increased production of pyrrolnitrin and other antifungal metabolites. To confirm its regulatory function, we constructed the vfr-knockout mutant $G05{\Delta}vfr$ and $G05{\Delta}phz{\Delta}prn::lacZ{\Delta}vfr$. By quantifying ${\beta}-galactosidase$ activities, we found that deletion of the vfr decreased the prn operon expression dramatically. Meanwhile, by quantifying pyrrolnitrin production in the mutant $G05{\Delta}vfr$, we found that deficiency of the Vfr caused decreased pyrrolnitrin production. However, production of phenazine-1-carboxylic acid was same to that in the wild-type strain G05. Taken together, Vfr is required for pyrrolnitrin but not for phenazine-1-carboxylic acid biosynthesis in P. chlororaphis G05.

• Journal of Plant Biotechnology
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• 제48권1호
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• pp.34-43
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• 2021

CRISPR/Cas9에 의한 유전자 교정 기술은 유용 형질을 갖는 작물 및 임목의 육성에 있어 널리 사용되고 있는 핵심 기술이다. 유전자 교정 임목 육성에는 아그로박테리움에 의한 형질전환 방법이 높은 효율로 시행된 연구가 많았고 따라서 형질전환에 사용된 플라스미드 서열이 식물 유전체 안에 존재한다는 문제가 남아 있었다. 본 연구에서는 CRISPR/Cas9을 사용하여 유전자 교정 임목을 육성하는 데 기존에 알려진 벡터 도입 기술이 아닌, 단일 가닥 가이드 RNA (sgRNA)와 Cas9 단백질을 혼합하여 만든 리보핵산단백질을 현사시나무 원형질체에 도입하는 방법을 기술하였다. 염 스트레스 내성 관련 인자 PagSAP1 유전자를 표적으로 하는 3종류의 sgRNA를 디자인하고, 각 sgRNA와 Cas9 단백질을 혼합하여 만든 리보핵산단백질을 원형질체에 도입하였다. 표적화 딥시퀀싱을 통해 리보핵산단백질 형성 시 sgRNA와 Cas9 단백질을 혼합하고 일정 시간 배양하여 안정화되는 시간이 필요한 것을 확인하였다. 또한 sgRNA3의 리보핵산단백질이 sgRNA1, sgRNA2의 리보핵산단백질보다 높은 교정 효율을 보이는 것을 확인하였다. 본 실험을 통해 리보핵산단백질을 이용한 유전자 교정 기술이 임목에도 적용될 수 있음이 확인되었고, 이는 외래 유전자 없이 유전자 교정 임목을 육성하는 데 활용할 수 있을 것으로 사료된다.

• Journal of Ginseng Research
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• 제45권4호
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• pp.465-472
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• 2021

Background: Ginseng can help regulate brain excitability, promote learning and memory, and resist cerebral ischemia in the central nervous system. Ginsenosides are the major effective compounds of Ginseng, but their protein targets in the brain have not been determined. Methods: We screened proteins that interact with the main components of ginseng (ginsenosides) by affinity chromatography and identified the 14-3-3 ζ protein as a potential target of ginsenosides in brain tissues. Results: Biolayer interferometry (BLI) analysis showed that 20(S)-protopanaxadiol (PPD), a ginseng saponin metabolite, exhibited the highest direct interaction to the 14-3-3 ζ protein. Subsequently, BLI kinetics analysis and isothermal titration calorimetry (ITC) assay showed that PPD specifically bound to the 14-3-3 ζ protein. The cocrystal structure of the 14-3-3 ζ protein-PPD complex showed that the main interactions occurred between the residues R56, R127, and Y128 of the 14-3-3 ζ protein and a portion of PPD. Moreover, mutating any of the above residues resulted in a significant decrease of affinity between PPD and the 14-3-3 ζ protein. Conclusion: Our results indicate the 14-3-3 ζ protein is the target of PPD, a ginsenoside metabolite. Crystallographic and mutagenesis studies suggest a direct interaction between PPD and the 14-3-3 ζ protein. This finding can help in the development of small-molecular compounds that bind to the 14-3-3 ζ protein on the basis of the structure of dammarane-type triterpenoid.

• 생명과학회지
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• 제32권9호
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• pp.729-733
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• 2022

수용성 단백질이 비정상적인 불용성 응집(aggregate)으로 전환되면 질환 등 여러 문제를 야기된다. 대장균 트립토판 중합효소 α 소단위체(αTS)는 가장 흔한 구조의 하나인 TIM 배럴 구조를 가지고 있다. 이전의 연구에서 불용성 응집이 일어나는 여러 잔기치환체(Y4C, S33L, P28L, P28S, G44S, D46N, P96L, P96S)를 얻었다. 본 연구에서는 높은 안정성과 도메인 협동성 성질을 보여주는 Y173F가 다른 잔기자리에 치환으로 유도된 응집 형성을 억제할 수 있는 지 여부를 조사했다. 8개 모두에서 억제효과를 보여 주었다. 단백질 분리가 가능한 P28L αTS를 분석한 결과, 이차구조함량의 감소, 안정성 감소, 소수성표면 증가 등의 구조변화특성을 보여주었다. Y173F가 첨가된 P28L/Y173F αTS은 야생형과 비슷한 구조로 회복되었다. 본 연구는 소수성 코아에 위치하는 Tyr¹⁷³ 잔기처럼 응집을 유도하는 여러 다른 잔기 자리를 보편적으로 억제하는 잔기가 존재할 수 있음을 시사해 준다.