• BMB Reports
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• 제40권6호
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• pp.1095-1099
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• 2007

Endonuclease G (EndoG) is a mitochondrial non-specific nuclease that is highly conserved among the eukaryotes. Although the precise role of EndoG in mitochondria is not yet known, the enzyme is released from the mitochondria and digests nuclear DNA during apoptosis in mammalian cells. Schizosaccharomyces pombe has an EndoG homolog Pnu1p (previously named SpNuc1) that is produced as a precursor protein with a mitochondrial targeting sequence. During the sorting into mitochondria the signal sequence is cleaved to yield the functionally active endonuclease. From the analogy to EndoG, active extramitochondrial Pnu1p may trigger cell killing by degrading nuclear DNA. Here, we tested this possibility by expressing a truncated Pnu1p lacking the signal sequence in the extramitochondrial region of pnu1-deleted cells. The truncated Pnu1p was localized in the cytosol and nuclei of yeast cells. And ectopic expression of active Pnu1p led to cell death with fragmentation of nuclear DNA. This suggests that the Pnu1p is possibly involved in a certain type of yeast cell death via DNA fragmentation. Although expression of human Bak in S. pombe was lethal, Pnu1p nuclease is not necessary for hBak-induced cell death.

• Journal of Plant Biotechnology
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• 제42권3호
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• pp.154-160
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• 2015

The plant breeding technology was developed with genetic engineering. Many researchers and breeders have turned from traditional breeding to molecular breeding. Genetically modified organisms (GMO) were developed via molecular breeding technology. Currently, molecular breeding technologies facilitate efficient plant breeding without introducing foreign genes, in virtue by of gene editing technology. Gene targeting (GT) via homologous recombination (HR) is one of the best gene editing methods available to modify specific DNA sequences in genomes. GT utilizes DNA repair pathways. Thus, DNA repair systems are controlled to enhance HR processing. Engineered sequence specific endonucleases were applied to improve GT efficiency. Engineered sequence specific endonucleases like the zinc finger nuclease (ZFN), TAL effector nuclease (TALEN), and CRISPR-Cas9 create DNA double-strand breaks (DSB) that can stimulate HR at a target site. RecQl4, Exo1 and Rad51 are effectors that enhance DSB repair via the HR pathway. This review focuses on recent developments in engineered sequence specific endonucleases and ways to improve the efficiency of GT via HR effectors in plants.

• Journal of Life Science
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• 제11권2호
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• pp.94-98
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• 2001

Type I signal peptidase cleaves the signal sequence from the amino terminus of membrane and secreted proteins afters these protein insert across the membrane. This enzyme serves as a potential target for the development of novel antibacterial agents due to its unique physiological and biochemical properties. Despite considerable research, the signal peptidase assay still remains improvement to provide further understanding of the mechanism and high-throughput inhibitor screening of this enzyme. In this paper, three known signal peptidase assays are tested with an E. coli D276A mutant signal peptidase to distinguish the sensitivity of each assays. In vitro assay using the procoat synthesized by in vitro transcription translation shows that the D276A signal peptidase I was inactive while in vivo processing of pro-OmpA expressed in the temperature-sensitive E. coli strain IT41 as well as in vitro assay using pro-OmpA nuclease A substrate show that D276A signal peptidase I has activity like wild-type signal peptidase. These results suggest that in vitro assay using the pro-OmpA nuclease A and in vivo pro-OmpA processing assay are more sensitive monitors than in vitro assay using the pro-coat. In conculsion, caution should be used when interpreting the in vitro results using the procoat.

• Bulletin of the Korean Chemical Society
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• 제35권10호
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• pp.3005-3008
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• 2014

Human ChlR1 protein (hChlR1), a member of the cohesion establishment factor family, plays an important role in the segregation of sister chromatids for maintenance of genome integrity. We previously reported that hChlR1 interacts with hFen1 and stimulates its nuclease activity on the flap-structured DNA substrate covered with RPA. To elucidate the relationship between hChlR1 and Okazaki fragment processing, the effect of hChlR1 on in vitro nuclease activities of hFen1 and hDna2 was examined. Independent of ATPase activity, hChlR1 stimulated endonuclease activity of hFen1 but not that of hDna2. Our findings suggest that the acceleration of Okazaki fragment processing near cohesions may aid in reducing the size of the replication machinery, thereby facilitating its entry through the cohesin ring.

• Journal of Microbiology and Biotechnology
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• 제25권8호
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• pp.1241-1245
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• 2015

The aim of the present work was to examine the putative promoter region of the operon ansPAB and to determine the general elements required for the regulation of transcriptional activity. The transcriptional start site of the ansPAB promoter was determined by using highresolution S1-nuclease mapping. Sequence analysis of this region showed -10 and -35 elements, which were consistent with consensus sequences for R. etli promoters that are recognized by the major form of RNA polymerase containing the σ⁷⁰ transcription factor. Mutation studies affecting several regions located upstream of the transcriptional start site confirmed the importance of these elements on transcriptional expression.

• Archives of Pharmacal Research
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• 제27권7호
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• pp.797-805
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• 2004

LPD vectors are non-viral vehicles for gene delivery comprised of polycation-condensed plasmid DNA and liposomes. Here, we described a novel anionic LPD formulation containing protamine-DNA complexes and pH sensitive liposomes composed of DOPE and cholesteryl hemisuccinate (Chems). Central composite design (CCD) was employed to optimize stable LPD formulation with small particle size. A three factor, five-level CCD design was used for the optimization procedure, with the weight ratio of protamine/DNA ($X_1$), the weight ratio of Chems/DNA ($X_2$) and the molar ratio of Chems/DOPE in the anionic liposomes ($X_3$) as the independent variables. LPD size ($Y_1$) and LPD protection efficiency against nuclease ($Y_2$) were response variables. Zeta potential determination was utilized to define the experimental design region. Based on experimental design, responses for the 15 formulations were obtained. Mathematical equations and response surface plots were used to relate the dependent and independent variables. The mathematical model predicted optimized $X_1-X_3$ levels that achieve the desired particle size and the protection efficiency against nuclease. According to these levels, an optimized LPD formulation was prepared, resulting in a particle size of 185.3 nm and protection efficiency of 80.22％.

• Laboraroty Animal Research
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• 제34권4호
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• pp.264-269
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• 2018

Cell cycle dysfunction can cause severe diseases, including neurodegenerative disease and cancer. Mutations in cyclin-dependent kinase inhibitors controlling the G1 phase of the cell cycle are prevalent in various cancers. Mice lacking the tumor suppressors $p16^{Ink4a}$ (Cdkn2a, cyclin-dependent kinase inhibitor 2a), $p19^{Arf}$ (an alternative reading frame product of Cdkn2a,), and $p27^{Kip1}$ (Cdkn1b, cyclin-dependent kinase inhibitor 1b) result in malignant progression of epithelial cancers, sarcomas, and melanomas, respectively. Here, we generated knockout mouse models for each of these three cyclin-dependent kinase inhibitors using engineered nucleases. The $p16^{Ink4a}$ and $p19^{Arf}$ knockout mice were generated via transcription activator-like effector nucleases (TALENs), and $p27^{Kip1}$ knockout mice via clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9). These gene editing technologies were targeted to the first exon of each gene, to induce frameshifts producing premature termination codons. Unlike preexisting embryonic stem cell-based knockout mice, our mouse models are free from selectable markers or other external gene insertions, permitting more precise study of cell cycle-related diseases without confounding influences of foreign DNA.

• 농업과학연구
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• 제47권4호
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• pp.903-920
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• 2020

Since its first demonstration as a practical genome editing tool in the early 2010s, the use of clustered regularly interspaced short palindromic repeat (CRISPR) along with the endonuclease Cas9 (CRISPR/Cas9) has become an essential choice for generating targeted mutations. Due to its relative simplicity and cost-effectiveness compared to other molecular scissors, i.e., zinc finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN), the CRISPR/Cas9 system has been shown to have a massive influence on genetic studies regardless of the biological kingdom. Although the system is in the process of being established, numerous protocols have already been released for the system and there have been various topics of CRISPR related papers published each year in ever-increasing manner. Here, we will briefly introduce CRISPR/Cas9 system and discuss the variants of the CRISPR system. Also, their applications to crop improvement will be dealt with mainly ornamental crops among horticultural crops other than Arabidopsis as a model plant. Finally, some issues on the barriers restraining the use of CRISPR system on floricultural crops, the prospect of CRISPR system as a DNA-free genome editing tool with efficient facilitators and finally, the future perspectives on the CRISPR system will be described.

• 한국원자력학회:학술대회논문집
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• 한국원자력학회 2004년도 추계학술발표회 발표논문집
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• pp.1126-1127
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• 2004

• 한국원자력학회:학술대회논문집
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• 한국원자력학회 2005년도 추계학술발표회
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• pp.811-812
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• 2005