• Bulletin of the Korean Chemical Society
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• v.34 no.4
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• pp.1232-1236
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• 2013

The Cu cation binding sites of $[Cu(I){\cdot}d(CpG){\cdot}d(CpG)-2H]^{-1}$ complex have been investigated to explain the $[Cu{\cdot}DNA]$ biological activity caused by the Cu association to DNA. The structure of $[Cu(I){\cdot}d(CpG){\cdot}d(CpG)-2H]^{-1}$ complex was investigated by electrospray ionization mass spectrometry (ESI-MS). The fragmentation patterns of $[Cu(I){\cdot}d(CpG){\cdot}d(CpG)-2H]^{-1}$ complex were analyzed by MS/MS spectra. In the MS/MS spectra of $[Cu(I){\cdot}d(CpG){\cdot}d(CpG)-2H]^{-1}$ complex, three fragment ions were observed with the loss of d(CpG), {d(CpG) + Cyt}, and {d(CpG) + Cyt + dR}. The Cu cation binds to d(CpG) mainly by substituting the $H^+$ of phosphate group. Simultaneously, the Cu cation prefers to bind to a guanine base rather than a cytosine base. Five possible geometries were considered in the attempt to optimize the $[Cu(I){\cdot}d(CpG){\cdot}d(CpG)-2H]^{-1}$ complex structure. The ab initio calculations were performed at B3LYP/6-31G(d) level.

• Journal of Microbiology and Biotechnology
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• v.15 no.5
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• pp.1022-1027
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• 2005

Based on plasmid display technology by the complexes of fusion protein and the encoding plasmid DNA, an in vitro selection method for high affinity DNA-binding protein was developed and experimentally demonstrated. The GAL4 DNA-binding domain (GAL4 DBD) was selected as a model DNA-binding protein, and enhanced green fluorescent protein (EGFP) was used as an expression reporter for the selection of target proteins. Error prone PCR was conducted to construct a mutant library of the model. Based on the affinity decrease with increased salt concentration, mutants of GAL4 DBD having high affinity were selected from the mutant protein library of protein-encoding plasmid complex by this method. Two mutants of (Lys33Glu, Arg123Lys, Ile127Lys) and (Ser47Pro, Ser85Pro) having high affinity were obtained from the first generation mutants. This method can be used for rapid in vitro selection of high affinity DNA-binding proteins, and has high potential for the screening of high affinity DNA-binding proteins in a sequence-specific manner.

• Ocean Science Journal
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• v.40 no.3
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• pp.155-166
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• 2005

New PCR primers (N=18) were designed for the isolation of complete SSU to LSU rDNA sequences from the dinoflagellate Alexandrium tamarense. Standard PCR, employing each primer set selected for amplifications of less than 1.5 kb, successfully amplified the expected rDNA regions of A. tamarense (Korean isolate, HY970328M). Complete SSU, LSU rDNAs and ITS sequences, including 5.8S rDNA, were recorded at 1,800 bp, 520 bp and 3,393 bp, respectively. The LSU rDNA sequence was the first report in Alexandrium genus. No intron was found in the LSU rRNA coding region. Twelve D-domains within the LSU rDNA were put together into 1,879 bp (44.4% G+C), and cores into 1514 bp (42.8% G+C). The core sequence was significantly different (0.0867 of genetic distance, 91% sequence similarity) in comparison with Prorocentrum micans (GenBank access. no. X16108). The D2 region was the longest in length (300 bp) and highly variable among the 12 D-domains. In a phylogenetic analysis using complete LSU rDNA sequences of a variety of phytoplankton, A. tamarense was clearly separated with high resolution against other species. The result suggests that the sequence may resolve the taxonomic ambiguities of Alexandrium genus, particularly of the tamarensis complex.

• Korean Journal of Plant Taxonomy
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• v.45 no.3
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• pp.243-253
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• 2015

Series Chinensis, Genus Iris, endemic to the far regions of East Asia, consists of four species and related varieties. This series is divided into two major groups (I. rossii and I. minutiaurea complex). In this study, the ITS region and matK gene sequences within nuclear ribosomal DNA and plastid DNA were analyzed in order to investigate the phylogenetic relationships among the I. minutiaurea complex (I. minutiaurea, I. odaesanensis, and I. koreana) and the taxonomic identities of a putative hybrid in Mt. Palgong. In the internal transcribed spacer (ITS1, 5.8S, and ITS2) region, a total of 106 cloned genomic sequences from three taxa were obtained to study the intragenomic polymorphisms of the ITS regions. Three taxa revealed high levels of intragenomic polymorphisms, indicative of incomplete nrDNA concerted evolution. This incomplete ITS concerted evolution in the series Chinensis may be linked to the recent species divergence and frequent interspecies hybridization of the series Chinensis. In the matK gene, three taxa were fairly separated by eleven variable sites. In eight individuals collected on Mt. Palgong, putative hybrids between I. odaesanensis and I. minutiaurea were clustered in the I. minutiaurea clade in the NJ (neighbor-joining) tree based on the matK gene. However, in the ITS tree, some of them were clustered in the I. odaesanensis clade and others were clustered in the I. minutiaurea clade. Therefore, the individuals on Mt. Palgong were formed by the hybridization between two taxa (I. odaesanensis and I. minutiaurea) and not through the lineage of I. koreana.

• Biomedical Science Letters
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• v.23 no.4
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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.

• Applied Biological Chemistry
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• v.48 no.2
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• pp.115-119
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• 2005

The catalytic hydrolysis reactivities of dinuclear nickel (II) complex, ${\mu}-aquapentaaqua[{\mu}-3,6-bis(6'-methyl-2'-pyridyl)pyridazine]chlorodinickel\;(II)$ trichloride trihydrate (APNT) for bis(p-nitrophenyl) phosphate (BNPP) as a DNA model compound were investigated. The dissociation constants of APNT were $pKa_1=7.9$ and $pKa_2=9.6$, respectively. The hydrolysis rate constant of BNPP compound by APNT was showed the rate enhancement of about 370,000 times in the case of none catalyst at pH 7.0 and $50^{\circ}C$. Based on the findings, we proposed the catalytic cycle for the hydrolysis of BNPP by APNT complex. The metal ions of dinuclear nickel (II) complex significantly enhance the transfer rate of phosphoryl group in the catalytic process and the water molecules as nucleophile and proton transfer agent act in different steps.

• Journal of the Korean Institute of Intelligent Systems
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• v.11 no.6
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• pp.538-542
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• 2001

DNA computing has been applied to the problem of getting an optimal soluting since Adleman's experiment. DNA computing uses strings with various length and four-type bases that makes more useful for finding a global optimal solutions of the complex multi-modal problems This paper presents DNA coding method finding optimal solution of the multi-modal function and compares the efficiency of this method with the genetic algorithms(GA). GA searches efffectively an optimal solution via the artificial evolution of individual group of binary string and DNA coding method uses DNA molecules and four-type bases denoted by the A(Ademine) C(Gytosine);G(Guanine)and T(Thymine). The selection, crossover, mutation operators are applied to both DNA coding algorithm and genetic algorithms and the comparison has been performed. The results show that the DNA based algorithm performs better than GA.

• Journal of Life Science
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• v.8 no.4
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• pp.400-409
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• 1998

Formation of adduct was studied in benzo(a)pyrene(BP)- and doxorubicin(Dx)-treated human NC-37 cells and isolated nuclei. Major adducts formed were determined by fluorescence absorption spectrophotometery and DNA-lin-ked protein assay. When isolated nuclei were exposed to carcinogens BP and DMBA, and anticancer drugs m-AMSA, ellipticine and Dx, varying degrees of adduct formation occured between DNA-protein complex and these drugs. When the mixture was centrifuged 1.7 M sucrose solution, binding BP and DMBA appeared to be similar between the sediment and the supernatant. When the sediment was centrifuged again with 0.35% polymin-P, the amount of BP bound was 2-fold greater in the protein(1077$\pm$55cpm) than in DNA fraction (470$\pm$20cpm), whereas that of DMBA was 1.6-fold greater in the DNA than in protein fraction. In the case of m-AMSA, ellipticine and Dx, the amount of binding was slightly greater in supernatant than in sediment in centrifugation with 1.7 M sucrose, and more than 3 times greater in the DNA- than in protein- fraction in centrifugation with 0.35% polymin P. DNA fractions which associated with a subset of nonhistone chromosomal protein were isolated from NC-37 cells exposed to $^{3}$H-BP and $^{14}$C-Dx. They were separated into two distince components DNA-S and DNA-P by centrifugation with 2M Nacl chromatin extraction. The results indicated that the amount of $^{3}$H-BP bound was 6.0-fold greater in DNA-P as compared with DNA-S, while that of $^{14}$C-Dx binding appreaed to be 6.2-fold greater in DNA-S than in DNA-P fraction. When $^{3}$H-BP binding wasdetermined in the presence of cold Dx, the amount of binding was reduced only in the DNA-P fraction, indicating that the interaction between DNA and protein is decreased. Gene expression by these drugs, BP treated cells were increased to compare with nomal cells but reduced by treatment with BP-Dx. These results suggest that the protein moiety which tightly bound to DNA-P fraction may play an important role in the regulation of gene expression.

• Journal of Microbiology and Biotechnology
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• v.27 no.6
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• pp.1098-1105
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• 2017

Vesicular stomatitis virus G glycoprotein (VSV-G) has been widely used for pseudotyping retroviral, lentiviral, and artificial viral vectors. The objective of this study was to establish a potential approach for large-scale production of VSV-G. To this end, VSV-G was cloned with an N-terminal His-tag into Pichia pastoris expression vector pPIC3.5K. Three clones ($Mut^s$) containing the VSV-G expression cassette were identified by PCR. All clones proliferated normally in expansion medium, whereas the proliferation was reduced significantly under induction conditions. VSV-G protein was detected in cell lysates by western blot analysis, and the highest expression level was observed at 96 h post induction. VSV-G could also be obtained from the condition medium of yeast protoplasts. Furthermore, VSV-G could be incorporated into Ad293 cells and was able to induce cell fusion, leading to the transfer of cytoplasmic protein. Finally, VSV-G-mediated DNA transfection was assayed by flow cytometry and luciferase measurement. Incubation of VSV-G lysate with the pGL3-control DNA complex increased the luciferase activity in Ad293 and HeLa cells by about 3-fold. Likewise, incubation of VSV-G lysate with the pCMV-DsRed DNA complex improved the transfection efficiency into Ad293 by 10% and into HeLa cells by about 1-fold. In conclusion, these results demonstrate that VSV-G could be produced from P. pastoris with biofunctionalities, demonstrating that large-scale production of the viral glycoprotein is feasible.

• Journal of Pharmaceutical Investigation
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• v.40 no.6
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• pp.357-362
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• 2010

To enhance therapeutic effects of insulin-sensitizing adipokine, ADN gene and potent agonists, rosiglitazone for the $PPAR{\gamma}$, cationic liposomes as non-viral vectors were formulated. The particle size and zeta potential of drug loaded and unloaded cationic liposomes were investigated. The complex formation between cationic liposomes and negatively charged plasmid DNA was confirmed and the protection from DNase was observed. In vitro transfection was investigated in HepG2, HeLa, and HEK293 cells by mRNA expression of ADN. Encapsulation efficacy of rosiglitazone-loaded liposomes was determined by UV detection. Particle sizes of cationic liposomes were in the range of 110-170 nm and those of rosiglitazone-loaded cationic liposomes were in the range of 130-180 nm, respectively. Gel retardation of complexes indicated that the complex was formed at weight ratios of cationic lipid to plasmid DNA higher than 20:1. Both complexes protected plasmid DNA from DNase either drug free or drug loading. Encapsulation efficiency of rosiglitazone-loaded emulsion was increased by drug dose. The mRNA expression levels of ADN were dose-dependently increased in cells transfected with plasmid DNA. Therefore, cationic liposomes could be potential co-delivery system for drug and gene.