• Korean Journal of Microbiology
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• 제23권1호
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• pp.13-19
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• 1985

Acc I endonuclease has been isolated from 300g (wet weight) cells of Acinetobacter calcoaceticus. The cells were broken by using French press at 20, 000p.s.i. After ammonium sulfate fractionation, the enzyme was further purified by heparin agarose, DEAE-sephades, Affi.-gel Blue, phosphocellulose, and hydroxylapatite column chromatography. The purified Acc I endonudlease has a single polypeptide species and its subunit molecular weight was 45,000 ${\pm}$ 1,000 daltons as judged by 10% SDS-polyacrylamide gel electrophoresis. The isolated enzyme was essentially free of contaminating nucleases as judged by homochromatography by using a $^{32}P-labeled$ oligonucleotide. The enzyme showed maximum activity at pH values between 8.0 and 11.0 and in the presence of $MgCl_2$. Acc I endonuclease was maximally active in the absence of NaCl and was completely inhibited at 200 mM NaCl.

• BMB Reports
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• 제49권4호
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• pp.226-231
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• 2016

Epstein Barr virus (EBV)-encoded nuclear antigen-1 (EBNA1) plays a pivotal in an EBV episome replication and persistence. Despite considerable attempts, there are no EBV drugs or vaccines. We attempted to eradicate EBV episomes by targeting EBNA1 using the transcription activator-like effector nucleases (TALEN) (E1TN). E1TN-mediated transient knockout (KO) of EBNA1 reduced EBNA1 expression, and caused significant loss of EBV genomes and progressive death of EBV-infected cells. Furthermore, when a mixture of EBV-infected Burkitt's lymphoma (BL) cells and EBV-negative BL cells was targeted by E1TN, EBV-negative cells were counter-selected while most EBV-infected cells died, further substantiating that EBNA1 KO caused selective death of EBV-infected cells. TALEN-mediated transient targeting of EBNA1 attenuated the growth of EBV-infected cells, implicating a possible therapeutic application of E1TN for EBV-associated disorders.

• BMB Reports
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• 제50권5호
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• pp.247-256
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• 2017

CRISPR/Cas9 is the latest tool introduced in the field of genome engineering and is so far the best genome-editing tool as compared to its precedents such as, meganucleases, zinc finger nucleases (ZFNs) and transcription activator-like effectors (TALENs). The simple design and assembly of the CRISPR/Cas9 system makes genome editing easy to perform as it uses small guide RNAs that correspond to their DNA targets for high efficiency editing. This has helped open the doors for multiplexible genome targeting in many species that were intractable using old genetic perturbation techniques. Currently, The CRISPR system is revolutionizing the way biological researches are conducted and paves a bright future not only in research but also in medicine and biotechnology. In this review, we evaluated the history, types and structure, the mechanism of action of CRISPR/Cas System. In particular, we focused on the application of this powerful tool in autophagy research.

• Genomics & Informatics
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• 제15권2호
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• pp.69-80
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• 2017

In this study, cytolethal distending toxin (CDT) producer isolates genome were compared with genome of pathogenic and commensal Escherichia coli strains. Conserved genomic signatures among different types of CDT producer E. coli strains were assessed. It was shown that they could be used as biomarkers for research purposes and clinical diagnosis by polymerase chain reaction, or in vaccine development. cdt genes and several other genetic biomarkers were identified as signature sequences in CDT producer strains. The identified signatures include several individual phage proteins (holins, nucleases, and terminases, and transferases) and multiple members of different protein families (the lambda family, phage-integrase family, phage-tail tape protein family, putative membrane proteins, regulatory proteins, restriction-modification system proteins, tail fiber-assembly proteins, base plate-assembly proteins, and other prophage tail-related proteins). In this study, a sporadic phylogenic pattern was demonstrated in the CDT-producing strains. In conclusion, conserved signature proteins in a wide range of pathogenic bacterial strains can potentially be used in modern vaccine-design strategies.

• Journal of Microbiology and Biotechnology
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• 제30권12호
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• pp.1919-1926
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• 2020

CRISPR interference (CRISPRi) has been developed as a transcriptional control tool by inactivating the DNA cleavage ability of Cas9 nucleases to produce dCas9 (deactivated Cas9), and leaving dCas9 the ability to specifically bind to the target DNA sequence. CRISPR/Cas9 technology has limitations in designing target-specific single-guide RNA (sgRNA) due to the dependence of protospacer adjacent motif (PAM) (5'-NGG) for binding target DNAs. Reportedly, Cas9-NG recognizing 5'-NG as the PAM sequence has been constructed by removing the dependence on the last base G of PAM through protein engineering of Cas9. In this study, a dCas9-NG protein was engineered by introducing two active site mutations in Cas9-NG, and its ability to regulate transcription was evaluated in the gal promoter in E. coli. Analysis of cell growth rate, D-galactose consumption rate, and gal transcripts confirmed that dCas9-NG can completely repress the promoter by recognizing DNA targets with PAM of 5'-NGG, NGA, NGC, NGT, and NAG. Our study showed possible PAM sequences for dCas9-NG and provided information on target-specific sgRNA design for regulation of both gene expression and cellular metabolism.

• Biomedical Science Letters
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• 제23권4호
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• pp.372-379
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• 2017

This study was investigated to test whether paternal DNA that was destined for degradation was properly licensed by testing for the presence of mini-chromosome maintenance protein (MCM) 7 and origin recognition complex (ORC) 2 in the paternal pronuclei. ORC2 is one of the first licensing protein to come on and MCM7 is one of the last licensing protein to come on. Zygotes were prepared by injection of control and treated sperm injection (ICSI). To control for DNA breakage, epididymal spermatozoa were treated with DNase I to fragment the DNA, then injected into oocytes. The presence of MCM7 and ORC2 in the pronuclei of mouse zygotes was tested by immunohistochemistry, just before the onset of DNA synthesis, at 5 h after fertilization, and after DNA synthesis began, at 9 h post fertilization. We found that in all cases, both MCM7 and ORC2 were present in both pronuclei at 5 h after sperm injection, just before DNA synthesis began. This indicates that no matter how extensive the DNA damage, recruitment of licensing proteins to the origins of replication was not inhibited. Sperm DNA fragmentation does not prevent licensing of DNA replication origins. Furthermore, the embryo recognizes DNA that is damaged by nucleases. Our data indicate that the one-cell embryo does harbor a mechanism to prevent the replication of severely damaged DNA from spermatozoa, even though the embryos do not undergo classical apoptosis.

• Molecules and Cells
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• 제40권11호
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• pp.823-827
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• 2017

Genome editing using programmable nucleases such as CRISPR/Cas9 or Cpf1 has emerged as powerful tools for gene knock-out or knock-in in various organisms. While most genetic diseases are caused by point mutations, these genome-editing approaches are inefficient in inducing single-nucleotide substitutions. Recently, Cas9-linked cytidine deaminases, named base editors (BEs), have been shown to convert cytidine to uridine efficiently, leading to targeted single-base pair substitutions in human cells and organisms. Here, we first report on the generation of Xenopus laevis mutants with targeted single-base pair substitutions using this RNA-guided programmable deaminase. Injection of base editor 3 (BE3) ribonucleoprotein targeting the tyrosinase (tyr) gene in early embryos can induce site-specific base conversions with the rates of up to 20.5%, resulting in oculocutaneous albinism phenotypes without off-target mutations. We further test this base-editing system by targeting the tp53 gene with the result that the expected single-base pair substitutions are observed at the target site. Collectively, these data establish that the programmable deaminases are efficient tools for creating targeted point mutations for human disease modeling in Xenopus.

• Asian-Australasian Journal of Animal Sciences
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• 제27권3호
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• pp.324-329
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• 2014

To facilitate the construction of genetically-modified pigs, we produced cloned embryos derived from porcine fibroblasts transfected with a pair of engineered zinc finger nuclease (ZFN) plasmids to create targeted mutations and enriched using a reporter plasmid system. The reporter expresses RFP and eGFP simultaneously when ZFN-mediated site-specific mutations occur. Thus, double positive cells ($RFP^+/eGFP^+$) were selected and used for somatic cell nuclear transfer. Two types of reporter based enrichment systems were used in this study; the cloned embryos derived from cells enriched using a magnetic sorting-based system showed better developmental competence than did those derived from cells enriched by flow cytometry. Mutated sequences, such as insertions, deletions, or substitutions, together with the wild-type sequence, were found in the cloned porcine blastocysts. Therefore, genetic mutations can be achieved in cloned porcine embryos reconstructed with ZFN-treated cells that were enriched by a reporter-based system.

• Korean Journal of Microbiology
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• 제25권3호
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• pp.180-183
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• 1987

Stu I, type II restriction endonuclease, has been purified to homogeneity from Streptomyces tubercidicus (ATCC 25502), and its catalytic properties have been studied. For the purification of Stu I endonuclease free of nonspecific nucleases, DEAE-Sephadex (A-50), QAE-Sephadex (A-50) and Heparin-agarose column chromatography have been performed after ammonium sulfate fractionation of the crude extract. The enzyme was further purified by gel filtration using Sephadex G-100 column to obtain homogeneous form of protein. The single polypeptide species of Stu I endonuclease has a subunit molecular weight of 34,000 $\pm$ 1,000 daltons as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Stu I endonuclease requires $Mg^{2+}$ ion for its activity and is maximally active at neutral pH (7.0-8.0) in the absence of NaCl.

• Journal of Applied Biological Chemistry
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• 제61권4호
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• pp.305-314
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• 2018

'New Breeding Techniques (NBTs)' have been one of hot issues, since their future will be affected profoundly by national as well as international regulatory landscapes. In this review, we compare characteristics of NBTs with conventional and genetic modification, and analyze genetically modified organism (GMO) regulatory systems in the context of possible regulation of NBTs. NBTs are very heterogeneous in terms of principles, methodologies, and final products. As Living Modified Organisms (LMO) is defined in the Cartagena Protocol on Biosafety (CPB) as an organism containing novel combination of genetic materials obtained by the use of modern biotechnology, CPB as well as other national legislations locate itself somewhere in the middle between product-based and process-based regulations. It is also noted that jurisdictions with regulatory systems more oriented to product-based one tend to be more productive and decide or may decide to exempt site-directed nucleases-1 from GMO regulation. In this context, Korean legislations are reviewed to clarify the commons and differences in GMO definitions. Act on Transboundary Movement of LMO Act, Food Sanitation Act and Agricultural and Fishery Products Quality Control Act are three major acts to regulate GMOs. It is noted that there are differences in the definition of LMO or GM food/products especially between the LMO Act and the Food Sanitation Act. Such differences may cause conflicts between Acts when policy-decision regarding the regulation of NBTs is made. Therefore, it is necessary to reorganize legislations before policies regarding the regulation of any techniques from biotechnology are made.