• Title/Summary/Keyword: DnaB

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Leucine Zipper as a Fine Tuner for the DNA Binding; Revisited with Molecular Dynamics Simulation of the Fos-Jun bZIP Complex

  • 최용훈;양철학;김현원;정선호
    • Bulletin of the Korean Chemical Society
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    • v.20 no.11
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    • pp.1319-1322
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    • 1999
  • Leucine zipper dynamically tunes the degree of bifurcation of the DNA binding segments in the basic region of the Fos-Jun bZIP complex. Molecular dynamics simulation indicated that site-specific mutagenesis of conserved leucine residues inside the leucine zipper domain caused the change of dynamic behavior of the basic region, and efficient DNA binding occurs only within a certain range of distance between the two DNA binding segments in the basic region. Distribution of α-helices in the hinge region is also suggested to influence the bifurcation of the DNA binding segments.

The Correlation between DNA Ploidy and the Clinicohistologic Findings in Colorectal Cancer (대장직장암에서 임상조직학적 소견과 DNA ploidy와의 상관관계)

  • Lee Suk Ho;Kim Hun Jung;Kim Woo Chul;Cho Young Kap;Loh John J K;Woo Ze Hong;Hwamg Tae Sook
    • Radiation Oncology Journal
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    • v.18 no.3
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    • pp.187-193
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    • 2000
  • Purpose : DNA ploidy pattern was shown to correlate with several clinicohistologic findings in several tumors. Aim of this study was to evaluate the correlation of the clinicohistologic findings in colorectal cancer and the failure pattern in rectosigmoid cancer with DNA ploidy. Materials and Methods DNA flow cytometry using the Hedley methods on paraffin embedded specimen from 117 patients with colorectal cancers after curative resection was peformed. We tried to find the correlation between DNA ploidy and various clinicohistologic findings. And then the correlation DNA ploidy and the failure pattern in 75 patients of rectosigmoid cancer was analized. Results : Forty samples (34.2%) from tumors gave aneuploidy histogram. There was no significant difference in the frequency of DNA aneuploidy in terms of age, sex, depth of invasion, location and Dukes stage. But there was a significant correlation between DNA ploidy and the failure rates in Dukes stage B rectosigmoid cancer (P=0.048). Conclusions : These findings suggest that DNA ploidy pattern shows the correlation with the treatment failure rates in Dukes stage B rectosigmoid, but not with many other clinicohistologic findings. However,more patients will be needed to disclose these findings.

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Phylogenetic Analysis of Korean Black Cattle Based on the Mitochondrial Cytochrome b Gene (mtDNA cytochrome b에 기초한 한국흑우의 계통유전학적 분석)

  • Kim, Jae-Hwan;Byun, Mi Jung;Kim, Myung-Jick;Suh, Sang Won;Kim, Young-Sin;Ko, Yeoung-Gyu;Kim, Sung Woo;Jung, Kyoung-Sub;Kim, Dong-Hun;Choi, Seong-Bok
    • Journal of Life Science
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    • v.23 no.1
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    • pp.24-30
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    • 2013
  • The purpose of this study was to identify genetic polymorphisms of the mitochondrial cytochrome b (mtDNA cyt b) gene in Korean black (KB) cattle breed and to analyze the genetic relationship between the KB and other breeds. We determined the complete sequence of the mtDNA cyt b gene in 38 KB cattle. We also analyzed their genetic diversity, and phylogenetic analysis was performed by comparison with Korean cattle (KC, called Hanwoo) and breeds from China and Japan. A nucleotide substitution was detected in the KB cattle, and two haplotypes were defined. In the neighbor-joining (NJ) tree, the haplotypes of KB were located in Bos taurus lineage with those of KC, Japanese black (JB), Yanbian and Zaosheng breeds. However, the haplotypes of Chinese breeds, excluding Yanbian and Zaosheng, were separated into B. taurus and B. indicus lineages. In the NJ tree of breeds based on Dxy genetic distances, Chinese breeds mixed with B. taurus and B. indicus lineages were located between B. indicus and B. taurus lineages. KB was contained within B. taurus lineage and was determined to be genetically more closely related to two Chinese (Yanbian and Zaosheng) breeds than to KC and JB. The haplotype distribution and the results of the phylogenetic analysis suggest that KB and KC have genetic differences in their mtDNA cyt b gene sequences.

Rapid and Unequivocal Identification Method for Event-specific Detection of Transgene Zygosity in Genetically Modified Chili Pepper

  • Kang, Seung-Won;Lee, Chul-Hee;Seo, Sang-Gyu;Han, Bal-Kum;Choi, Hyung-Seok;Kim, Sun-Hyung;Harn, Chee-Hark;Lee, Gung-Pyo
    • Horticultural Science & Technology
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    • v.29 no.2
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    • pp.123-129
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    • 2011
  • To identify unintended vertical gene-transfer rates from the developed transgenic plants, rapid and unequivocal techniques are needed to identify event-specific markers based on flanking sequences around the transgene and to distinguish zygosity such as homo- and hetero-zygosity. To facilitate evaluation of zygosity, a polymerase chain reaction technique was used to analyze a transgenic pepper line B20 (homozygote), P915 wild type (null zygote), and their F1 hybrids, which were used as transgene contaminated plants. First, we sequenced the 3'-flanking region of the T-DNA (1,277 bp) in the transgenic pepper event B20. Based on sequence information for the 3'- and 5'-flanking region of T-DNA provided in a previous study, a primer pair was designed to amplify full length T-DNA in B20. We successfully amplified the full length T-DNA containing 986 bp from the flanking regions of B20. In addition, a 1,040 bp PCR product, which was where the T-DNA was inserted, was amplified from P915. Finally, both full length T-DNA and the 1,040 bp fragment were simultaneously amplified in the F1 hybrids; P915 ${\times}$ B20, Pungchon ${\times}$ B20, Gumtap ${\times}$ B20. In the present study, we were able to identify zygosity among homozygous transgenic event B20, its wild type P915, and hemizygous F1 hybrids. Therefore, this novel zygosity identification technique, which is based on PCR, can be effectively used to examine gene flow for transgenic pepper event B20.

Molecular Cloning of Chitosanase Gene and Quantitative Production of Chitosan Oligomer (키토사네이즈 유전자의 클로닝과 키토산 올리고머의 정량적 생산)

  • 박유미;장혜란;허태린;김사열
    • Microbiology and Biotechnology Letters
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    • v.32 no.1
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    • pp.16-21
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    • 2004
  • Six bacterial strains which formed large halo on chitosan-containing agar plate were isolated from beach mud and crabs at South coast of Korean peninsula. They were designated as Bacillus cereus KNUC51, B. cereus KNUC52, B. cereus KNUC53, B. cereus KNUC54, B. cereus KNUC55, and Paenibacillus favisporus KNUC56 by analysing their morphologies and 16S rDNA sequences. Chitosanase activities of all isolates were similar to that of B. subtilis 168. To enhance the activity of chitosanase, a powerful mutagen, MNNG was treated for P favisporus KNUC56. Three mutants showed higher activity of chitosanase than that of the original strain. The DNA fragments containing chitosanase gene from B. cereus sources were cloned, sequenced, and their deduced amino acid sequence analysis showed over 93% homologies with that of the known B. cereus ATCC14579. Extracellular sample from the isolates was incubated in proper reaction mixture including chitosan for 5 minutes at $37^{\circ}C$ to produce 3-10 chitosan oligomers which has been known to be active for clinical agents and agronomical agents.

Cloning, Expression, and Characterization of a Family B-Type DNA Polymerase from the Hyperthermophilic Crenarchaeon Pyrobaculum arsenaticum and Its Application to PCR

  • SHIN HEA-JIN;LEE SUNG-KYOUNG;CHOI JEONG JIN;KOH SUK-HOON;LEE JUNG-HYUN;KIM SANG-JIN;KWON SUK-TAE
    • Journal of Microbiology and Biotechnology
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    • v.15 no.6
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    • pp.1359-1367
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    • 2005
  • The gene encoding Pyrobaculum arsenaticum DNA polymerase (Par DNA polymerase) was cloned and sequenced. The gene consists of 2,361 bp coding for a protein with 786 amino acid residues. The deduced amino acid sequence of Par DNA polymerase showed a high similarity to archaeal family B-type DNA polymerases (Group I), and contained all of the motifs conserved in the family B-type DNA polymerases for $3'{\rightarrow}5'$ exonuclease and polymerase activities. The Par DNA polymerase gene was expressed under the control of the T7lac promoter on the expression vector pET-22b(+) in Escherichia coli BL21-CodonPlus(DE3)-RP. The expressed enzyme was purified by heat treatment, and Cibacron blue 3GA and $Hirap^{TM}$ Heparin HP column chromatographies. The optimum pH of the purified enzyme was 7.5. The enzyme activity was activated by divalent cations, and was inhibited by EDTA and monovalent cations. The half-life of the enzyme at $95^{\circ}C$ was 6 h. Par DNA polymerase possessed associated $3'{\rightarrow}5'$ proofreading exonuclease activity, which is consistent with its deduced amino acid sequence. PCR experiment with Par DNA polymerase showed an amplified product, indicating that this enzyme might be useful in DNA amplification and PCR-based applications.

NMR Study on the Preferential Binding of the Zα Domain of Human ADAR1 to CG-repeat DNA Duplex

  • Lee, Ae-Ree;Choi, Seo-Ree;Seo, Yeo-Jin;Lee, Joon-Hwa
    • Journal of the Korean Magnetic Resonance Society
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    • v.21 no.3
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    • pp.90-95
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    • 2017
  • The Z-DNA domain of human ADAR1 ($Z{\alpha}_{ADAR1}$) produces B-Z junction DNA through preferential binding to the CG-repeat segment and destabilizing the neighboring AT-rich region. However, this study could not answer the question of how many base-pairs in AT-rich region are destabilized by binding of $Z{\alpha}_{ADAR1}$. Thus, we have performed NMR experiments of $Z{\alpha}_{ADAR1}$ to the longer DNA duplex containing an 8-base-paired (8-bp) CG-repeat segment and a 12-bp AT-rich region. This study revealed that $Z{\alpha}_{ADAR1}$ preferentially binds to the CG-repeat segment rather than AT-rich region in a long DNA and then destabilizes at least 6 base-pairs in the neighboring AT-rich region for efficient B-Z transition of the CG-repeat segment.

CpG-DNA induces bacteria-reactive IgM enhancing phagocytic activity against Staphylococcus aureus infection

  • Kim, Te Ha;Kim, Dongbum;Lee, Heesu;Kwak, Min Hyung;Park, Sangkyu;Lee, Younghee;Kwon, Hyung-Joo
    • BMB Reports
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    • v.52 no.11
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    • pp.635-640
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    • 2019
  • CpG-DNA triggers the proliferation and differentiation of B cells which results in the increased production of antibodies. The presence of bacteria-reactive IgM in normal serum was reported; however, the relevance of CpG-DNA with the production of bacteria-reactive IgM has not been investigated. Here, we proved the function of CpG-DNA for the production of bacteria-reactive IgM. CpG-DNA administration led to increased production of bacteria-reactive IgM both in the peritoneal fluid and serum through TLR9 signaling pathway. When we stimulated B cells with CpG-DNA, production of bacteria-reactive IgM was reproduced in vitro. We established a bacteria-reactive monoclonal IgM antibody using CpG-DNA stimulated-peritoneal B cells. The monoclonal IgM antibody enhanced the phagocytic activity of RAW 264.7 cells against S. aureus MW2 infection. Therefore, we suggest that CpG-DNA enhances the antibacterial activity of the immune system by triggering the production of bacteria-reactive IgM. We also suggest the possible application of the antibodies for the treatment of antibiotics-resistant bacterial infections.

Hepatitis B Virus X Protein Stimulates Virus Replication Via DNA Methylation of the C-1619 in Covalently Closed Circular DNA

  • Lee, Hyehyeon;Jeong, Hyerin;Lee, Sun Young;Kim, Soo Shin;Jang, Kyung Lib
    • Molecules and Cells
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    • v.42 no.1
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    • pp.67-78
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    • 2019
  • Methylation of HBV cccDNA has been detected in vivo and in vitro; however, the mechanism and its effects on HBV replication remain unclear. HBx derived from a 1.2-mer HBV replicon upregulated protein levels and enzyme activities of DNA methyltransferase 1 (DNMT1), 3a, and 3b, resulting in methylation of the negative regulatory region (NRE) in cccDNA, while none of these effects were observed with an HBx-null mutant. The HBx-positive HBV cccDNA expressed higher levels of HBc and produced about 4-fold higher levels of HBV particles than those from the HBx-null counterpart. For these effects, HBx interrupted the action of NRE binding protein via methylation of the C-1619 within NRE, resulting in activation of the core promoter. Treatment with 5-Aza-2′dC or DNMT1 knock-down drastically impaired the ability of HBx to activate the core promoter and stimulate HBV replication in 1.2-mer HBV replicon and in vitro infection systems, indicating the positive role of HBx-mediated cccDNA methylation in HBV replication.

Comparison of the Cell Surface Barrier and Enzymatic Modification System in Brevibacterium flavum and B. Lactofermentum

  • Jang Ki-Hyo;Britz Margaret L.
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.10 no.3
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    • pp.225-229
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    • 2005
  • To investigate impediments to plasmid transformation in Brevibacterium flavum BF4 and B. lactofermentum BL1, cell surface barriers were determined by measuring growth inhibition whilst enzymatic barriers were determined by comparing DNA methylation properties. B. lactofermentum was more sensitive to growth inhibition by glycine than B. flavum. Release of cellular proteins during sonication was more rapid for B. lactofermentum than for B. flavum. Plasmid DNA (pCSL 17) isolated from B. flavum transformed recipient $McrBC^+$ strains of Escherichia coli with lower efficiency than $McrBC^-$. McrBC digestion of this DNA confirmed that B. flavum contain methylated cytidines in the target sequence of McrBc sequences but B. lactofermentum contained a different methylation pattern. DNA derived from the B. lactofermentum transformed recipient $EcoKR^+$ strains of E. coli with lower efficiency than $EcoKR^-$, indicating the presence of methylated adenosines in the target sequence of EcoK sequences. The present data describe the differences in the physical and enzymatic barriers between two species of corynebacteria and also provide some insight into the successful foreign gene expression in corynebacteria.