• Korean Journal of Microbiology
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• v.28 no.1
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• pp.19-26
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• 1990

To examine the effects of sequence variations near the transcriptional start site on the rate of formation of the open complexes at bacteriophage $\lambda P_{R}$ promoter, two mutant promoters were created by site-specific mutagenesis using synthetic oligonucleotides. Mutant I coatains changes at positions -3 and -4 from TT to CC, thus having a 6-bp long G/C stretch between -10 region and transciptional start site (+1). Mutant II has changes at positions -5 and -6 from GG to AA, thereby having a 9-bp long A/T stretch between positions -11 and -3. Selective filter binding assays were performed to measure the rate of formation of the open complexes between the wild-type or two mutant $P_{R}$ promoters on 664 bp fragments and E. coli RNA polymerase at two temperatures. At 37.deg.C, the wild-type and two mutants showed similar rates for the formation of open complex. The second order rate constant $k_{a}$ and $\tau _{int}$, as determined from the .tau.-plot analysis, were $(6.0\pm0.4)\times10^{6}M^{-1}sec^{-1}$ and $11\pm5$sec, respectively. At 18.deg.C, however, the wild-type and two mutant promoters showed differences in the kinetic parameters. k for the wild-type promoter was (2.2$\pm$0.1)\times 10^{6}M^{-1}sec^{-1}$ and $\tau _{int}$ was 76$\pm$sec. Mutant I and II exhibited differences mainly in the rate of isomerization ($\tau_{int,I}=91\pm$10 sec, int,II=34$\pm$ sec), whereas the second order rate constant $k_{a}$ was similar to the wild type value. This result implies that at $18^{\circ}C$, the isomerization rate is determined by both protein conformational change and DNA melting, which are separable kinetically according to the 3-step mechanism of Roe et al.(1984,1985), and that the base changes affected mainly the rate of DNA melting as predicted.lting as predicted.

• Macromolecular Research
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• v.17 no.4
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• pp.245-249
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• 2009

A fluorescing, copolymer(Q)-bearing, quaternary ammonium pendant was mixed with excess natural salmon sperm DNA with a molecular weight of $1.3{\times}10^6$(2,000 base pairs) to afford highly fluorescing, complex mixtures. The fluorescence life-time of the polymer Q was greatly increased when mixed with DNA: for the mixture of Q:DNA=1:750 the fast and slow decay lifetimes increased from ca. 10 to 100 ps and from 20 ps to ca. 1 ns, respectively. The enhanced fluorescence of the mixtures was ascribed to efficient compartmentalization and reduced conformational relaxation of the polymer Q by complexation with excess DNA.

• BMB Reports
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• v.34 no.1
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• pp.1-7
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• 2001

Structural and physical properties of type wild type and various selected mutants of the vnd/NK-2 homeodomain, the protein product of the homeobox, and the implication in genetic disease are reviewed. The structure, dynamics and thermodynamics have been Investigated by NMR and by calorimetry. The interactions responsible for the nucleotide sequence-specific binding of the homeodomain to its consensus DNA binding site have been identified. There is a strong correlation between significant structural alterations within the homeodomain or its DNA complex and the appearance of genetic disease. Mutations in positions known to be important in genetic disease have been examined carefully For example, mutation of position 52 of vnd/NK-2 results in a significant structural modification and mutation of position 54 alters the DNA binding specificity and amity The $^{15}N$ relaxation behavior and heteronuclear Overhauser effect data was used to characterize and describe the protein backbone dynamics. These studies were carried out on the wild type and the double mutant proteins both in the free and in the DNA bound states. Finally, the thermodynamic properties associated with DNA binding are described for the vnd/NK-2 homeodomain. These thermodynamic measurements reinforce the hypothesis that water structure around a protein and around DNA significantly contribute to the protein-DNA binding behavior. The results, taken together, demonstrate that structure and dynamic studies of proteins combined with thermodynamic measurements provide a significantly more complete picture of the solution behavior than the individual studies.

• BMB Reports
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• v.42 no.1
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• pp.53-58
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• 2009

The methyl-directed mismatch repair (MMR) mechanism has been extensively studied in vitro and in vivo, but one of the difficulties in determining the biological relationships between the MMR-related proteins is the tendency of MutL to self-aggregate. The properties of a stable MutL homologue were investigated using a thermostable MutL (TmL) from Thermotoga maritima MSB8 and whose size exclusion chromatographic and crosslinking analyses were compatible with a dimeric form of TmL. TmL underwent conformational changes in the presence of nucleotides and single-stranded DNA (ssDNA) with ATP binding not requiring ssDNA binding activity of TmL, while ADPnP-stimulated TmL showed a high ssDNA binding affinity. Finally, TmL interacted with the T. maritima MutS (TmS), increasing the affinity of TmS to mismatched DNA base pairs and suggesting that the role of TmL in the formation of a mismatched DNA-TmS complex may be a pivotal observation for the study of the initial MMR system.

• Reproductive and Developmental Biology
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• v.34 no.4
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• pp.283-288
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• 2010

During normal early embryonic development in mammals, the global pattern of genomic DNA methylation undergoes marked. changes. The level of methylation is high in male and female gametes. Thus, we cloned the cDNA of the porcine DNA methyltransferase 1 (Dnmt1) gene to promote the efficiency of the generation of porcine clones. In this study, porcine Dnmt1 cDNA was sequenced, and Dnmt1 mRNA expression was detected by reverse transcription-polymerase reaction (RT-PCR) in porcine tissues during embryonic development. The porcine Dnmt1 cDNA sequence showed more homology with that of bovine than human, mouse, and rat. The complete sequence of porcine Dnmt1 cDNA was 4,774-bp long and consisted of an open reading frame encoding a protein of 1611 amino acids. The amino acid sequence of porcine DNMT1 showed significant homology with those of bovine (91%), human (88%), rat (76%), and mouse (75%) Dnmt1. The expression of porcine Dnmt1 mRNA was detected during porcine embryogenesis. The mRNA was detected at stages of porcine preimplantation development (1-cell, 2-cell, 4-cell, 8-cell, morula, and blastocyst stages). It was also abundantly expressed in tissues (lung, ovary, kidney and somatic cells). Further investigations are necessary to understand the complex links between methyltransferase 1 and the transcriptional activity in cloned porcine tissues.

• Bulletin of the Korean Chemical Society
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• v.32 no.3
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• pp.947-955
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• 2011

The complexes [Pd(bpy)(Hex-dtc)]$NO_3$ and [Pt(bpy)(Hex-dtc)]$NO_3$ (bpy is 2,2'-bipyridine and Hex-dtc is hexyldithiocarbamato ligands) were synthesized and characterized by elemental analysis and spectroscopic studies. The cytotoxicity assay of the complexes has been performed on chronic myelogenous leukemia cell line, K562, at micromolar concentration. Both complexes showed cytotoxic activity far better than that of cisplatin under the same experimental conditions. The binding parameters of the complexes with calf thymus DNA (CT-DNA) was investigated using UV-visible and fluorescence techniques. They show the ability of cooperatively intercalating in CT-DNA. Gel filtration studies demonstrated that platinum complex could cleave the DNA. In the interaction studies between the Pd(II) and Pt(II) complexes with CT-DNA, several binding and thermodynamic parameters have been determined, which may provide deeper insights into the mechanism of action of these types of complexes with nucleic acids.

• Journal of Microbiology and Biotechnology
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• v.31 no.9
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• pp.1323-1329
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• 2021

Micro-scale magnetic beads are widely used for isolation of proteins, DNA, and cells, leading to the development of in vitro diagnostics. Efficient isolation of target biomolecules is one of the keys to developing a simple and rapid point-of-care diagnostic. A zinc finger protein (ZFP) is a double-stranded (ds) DNA-binding domain, providing a useful scaffold for direct reading of the sequence information. Here, we utilized two engineered ZFPs (Stx2-268 and SEB-435) to detect the Shiga toxin (stx2) gene and the staphylococcal enterotoxin B (seb) gene present in foodborne pathogens, Escherichia coli O157 and Staphylococcus aureus, respectively. Engineered ZFPs are immobilized on a paramagnetic bead as a detection platform to efficiently isolate the target dsDNA-ZFP bound complex. The small paramagnetic beads provide a high surface area to volume ratio, allowing more ZFPs to be immobilized on the beads, which leads to increased target DNA detection. The fluorescence signal was measured upon ZFP binding to fluorophore-labeled target dsDNA. In this study, our system provided a detection limit of ≤ 60 fmol and demonstrated high specificity with multiplexing capability, suggesting a potential for development into a simple and reliable diagnostic for detecting multiple pathogens without target amplification.

• Genomics & Informatics
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• v.17 no.1
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• pp.11.1-11.7
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• 2019

Athletic performance is a complex multifactorial trait involving genetic and environmental factors. The heritability of an athlete status was reported to be about 70% in a twin study, and at least 155 genetic markers are known to be related with athlete status. Mitochondrial DNA (mtDNA) encodes essential proteins for oxidative phosphorylation, which is related to aerobic capacity. Thus, mtDNA is a candidate marker for determining physical performance. Recent studies have suggested that polymorphisms of mtDNA are associated with athlete status and/or physical performance in various populations. Therefore, we analyzed mtDNA haplogroups to assess their association with the physical performance of Korean population. The 20 mtDNA haplogroups were determined using the SNaPshot assay. Our result showed a significant association of the haplogroup F with athlete status (odds ratio, 3.04; 95% confidence interval, 1.094 to 8.464; p = 0.012). Athletes with haplogroup F ($60.64{\pm}3.04$) also demonstrated a higher Sargent jump than athletes with other haplogroups ($54.28{\pm}1.23$) (p = 0.041). Thus, our data imply that haplogroup F may play a crucial role in the physical performance of Korean athletes. Functional studies with larger sample sizes are necessary to further substantiate these findings.

• Proceedings of the Botanical Society of Korea Conference
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• 1999.08a
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• pp.55-67
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• 1999

A puc -deleted cell of Rhodobacter sphaeroides grows with a doubling time longer than 160 h under the light-limiting photoheterotrophic ( 3 Watts [W]/㎡) conditions due to an absence of the peripheral light-harvesting B800-850 complex. A spontaneous fast-growing mutant, R.sphaeroides SK101 was ioslate dto have∼40-h doubling at 3 Watts/㎡, while the growth of the mutant was not distinguished from its parental strain during both aerobic and light-saturating photoheterotrphic (10W/㎡) growth. The B875 complex of SK101 under the light-limiting conditions was elevated by 20 to 30% compared with that of the puc -deleted cell, reflecting parallel increase of bacteriochlorophyll and carotenoid contents of the mutant. The formation of B875 complex of SK101 under the anaerobic dark conditions with dimethylsulfoxide was the same as that of the puc-deleted cell. suggesting that the mutation of SK101 result in the altered control of B875 complex formation by light. When puc is restored in SK101 , it is not B875 complex but B800-850 complex which formation is elevated. The mutation of SK101 affected the bchF transcription most drastically to show two to tenfold increase during both aerobic and photoheterotrophic growth. The mutated phenotype of SK101 was complemented with pW2, which contains approximately 100-kb HNA of the photosynthetic gene clusters. The complementing DNA was narrowed down to a 1.1-kb DNA containing orfQ and pufKBA . The mutation of SK101 appeared to be exerted through the mutation of the orfQ gene encoding a putative bacteriochlorophyll -mobilizing protein.

• BMB Reports
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• v.29 no.6
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• pp.569-573
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• 1996

Clinical strains of Mycobacterium tuberculosis, M. terrae complex, M. gordonae, M. avium-intracellulae complex, and M. fortuitum from Korean patients were isolated and analyzed by comparing large restriction fragment (LRF) patterns produced by digestion of genomic DNA with infrequent-cutting endonucleases like AsnI and XbaI. and pulsed-field gel electrophoresis (PFGE). Three M. tuberculosis, two M. terrae complex, two M. gordonae, two M. avium-intracellulae complex, and two M. fortuitum strains were compared by using AsnI and XbaI. and this allowed easy visual separation of all epidemiologically unrelated strains. PFGE exhibits different DNA restriction patterns which are easy to compare. Genome size of the strains roughly ranged from 3020 to 3335 kb. The LRF patterns are useful for epidemiologic studies of tuberculosis with regard to drug resistance.