• BMB Reports
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• v.57 no.1
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• pp.30-39
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• 2024

Directed evolution (DE) of desired locus by targeted random mutagenesis (TRM) tools is a powerful approach for generating genetic variations with novel or improved functions, particularly in complex genomes. TRM-based DE involves developing a mutant library of targeted DNA sequences and screening the variants for the desired properties. However, DE methods have for a long time been confined to bacteria and yeasts. Lately, CRISPR/Cas and DNA deaminase-based tools that circumvent enduring barriers such as longer life cycle, small library sizes, and low mutation rates have been developed to facilitate DE in native genetic environments of multicellular organisms. Notably, deaminase-based base editing-TRM (BE-TRM) tools have greatly expanded the scope and efficiency of DE schemes by enabling base substitutions and randomization of targeted DNA sequences. BE-TRM tools provide a robust platform for the continuous molecular evolution of desired proteins, metabolic pathway engineering, creation of a mutant library of desired locus to evolve novel functions, and other applications, such as predicting mutants conferring antibiotic resistance. This review provides timely updates on the recent advances in BE-TRM tools for DE, their applications in biology, and future directions for further improvements.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.445-449
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• 2003

As for the purpose, we first introduce an random mutation into wild-type gene to expand a mutation space, and then further recombine the mutant genes by staggered extension process PCR. As a result, we obtained the best clones 6-52 that showed a high activity and stability, from a round of error prone and staggered extension process PCR. The purified enzyme showed a similar pH stability to the wild-type enzyme and reveal a slightly high optimum pH at 12. In the optimum temperature, an identical dependency was also showed and a quite high stability in the thermal stability was obtained. Along with this, the enzyme was also stable at a reaction that supplement with a 15 % of ethanol as an additive. The addition of other solvents and surfactants did not improve the reaction and thus resulted in a similar profile to those of wild-type enzyme. The specific activity on the target compound rac-ketoprofen ethyl ester was calculated to be about 85, 000 unit, and the kinetic constants Km and Vmax were determined to be 0.2 mM and 90 mM/mg-protein/min respectively. The deduced amino acid alignment with the wild type enzyme revealed five mutations at L120P, I208V, T249A, D287H and T357A. Based on these observations, the site directed mutagenesis to delineate the mutagenic effect is under progress.

• Environmental Mutagens and Carcinogens
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• v.22 no.2
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• pp.106-111
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• 2002

AG6O, the complex of acriflavine and guanosine, has been shown to possess the synergistic antitumorigenic activity in the previous paper (J. Pharm. Pharmacol. 1997, 49:216). In this study, we have investigated the genotoxic properties of AG60 using in vitro and in vivo system such as Ames bacterial reversion test, chromosomal aberration assay and micronucleus assay. In Ames reverse mutation test, AG60 treatment at the dose range up to 250 $\mu\textrm{g}$/plate caused the dose-independent random induction of the mutagenic colony formation in S. typhimurium TA98, TA100, TA1537, and E. coli WP2uvrA, while any mutagenic effect of AG60 wasn't observed in S. typhimurium TA1535. Any significant chromosomal aberration wasn't observed in chinese hamster lung (CHL) fibroblast cells incubated with PBS or AG60 at the concentrations of 2.5, 5, 10 $\mu\textrm{g}$/$m\ell$ for 24 hours without but even with 59 metabolic activation system for 6 hours. In vivo ICR mice, the intramuscular injection of AG60 at the doses of 7.15, 14.3, and 28.6 mg/kg did not induce the frequency of micronucleus formation. However, mitomycin C, as one of the positive controls at the dose of 2 mg/kg caused the 8.4% induction in the frequency of micronucleus and 24% increase in the chromosomal aberration.

• Management Science and Financial Engineering
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• v.15 no.1
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• pp.51-69
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• 2009

A network model and a Genetic Algorithm (GA) is proposed to solve the simultaneous estimation of the trip distribution and traffic assignment from traffic counts in the congested networks in a logit-based Stochastic User Equilibrium (SUE). The model is formulated as a problem of minimizing a non-linear objective function with the linear constraints. In the model, the flow-conservation constraints are utilized to restrict the solution space and to force the link flows become consistent to the traffic counts. The objective of the model is to minimize the discrepancies between two sets of link flows. One is the set of link flows satisfying the constraints of flow-conservation, trip production from origin, trip attraction to destination and traffic counts at observed links. The other is the set of link flows those are estimated through the trip distribution and traffic assignment using the path flow estimator in the logit-based SUE. In the proposed GA, a chromosome is defined as a real vector representing a set of Origin-Destination Matrix (ODM), link flows and route-choice dispersion coefficient. Each chromosome is evaluated by the corresponding discrepancies. The population of the chromosome is evolved by the concurrent simplex crossover and random mutation. To maintain the feasibility of solutions, a bounded vector shipment technique is used during the crossover and mutation.

• Journal of Microbiology and Biotechnology
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• v.28 no.6
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• pp.1030-1036
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• 2018

Bacillus strains produce various types of antibiotics, and random mutagenesis has traditionally been used to overproduce these natural metabolites. However, this method leads to the accumulation of unwanted mutations in the genome. Here, we rationally designed a single nucleotide substitution in the degU gene to generate a B. subtilis strain displaying increased plipastatin production in a foreign DNA-free manner. The mutant strain (BS1028u) showed improved antifungal activity against Pythium ultimum. Notably, pps operon deletion in BS1028u resulted in complete loss of antifungal activity, suggesting that the antifungal activity strongly depends on the expression of the pps operon. Quantitative real-time PCR and lacZ assays showed that the point mutation resulted in 2-fold increased pps operon expression, which caused the increase in antifungal activity. Likewise, commercial Bacillus strains can be improved to display higher antifungal activity by rationally designed simple modifications of their genome, rendering them more efficient biocontrol agents.

• Plant Breeding and Biotechnology
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• v.6 no.4
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• pp.426-433
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• 2018

Plant tissue culture is a technique that has invariably been used for various purposes such as obtaining transgenic plants for crop improvement or functional analysis of genes. However, this process can be associated with a variety of genetic and epigenetic instabilities in regenerated plants, termed as somaclonal variation. In this study, we investigated mutation spectrum, chromosomal distributions of nucleotide substitution types of single-nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) by whole genome re-sequencing between Dongjin and Nipponbare along with regenerated plants of Dongjin from different induction periods. Results indicated that molecular spectrum of mutations in regenerated rice against Dongjin genome ranged from $9.14{\times}10^{-5}$ to $1.37{\times}10^{-4}$ during one- to three-month callus inductions, while natural mutation rate between Dongjin and Nipponbare genomes was $6.97{\times}10^{-4}$. Non-random chromosome distribution of SNP and InDel was observed in both regenerants and Dongjin genomes, with the highest densities on chromosome 11. The transition to transversion ratio was 2.25 in common SNPs of regenerants against Dongjin genome with the highest C/T transition frequency, which was similar to that of Dongjin against Nipponbare genome.

• Journal of Life Science
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• v.22 no.1
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• pp.7-15
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• 2012

A polyguluronate-specific lyase from Streptomyces sp. strain M3 has been previously cloned and characterized. In this study, the M3 alginate lyase gene in the pColdI vector was mutated by site-directed mutagenesis and random mutagenesis to enhance the alginate degrading activity. Six mutants were obtained: Ser25Arg, Phe99Leu, Asp142Asn, Val163Ala, Lys191Glu, and Gly194Cys. Phe99Leu and Lys191Glu mutants completely lost their alginate lyase activity, whereas the alginate degrading activity of Gly194Cys mutant increased by nearly 10 fold. The 3-D protein structure of M3 alginate lyase, which was constructed using the Swiss-Model automodeler, was also compared to the crystal structure of another alginate lyase. A mutated glycine residue was positioned between Gly193 and Tyr195 of the C-terminal conserved sequence, YFKAGXYXQ. A phenylalanine residue (at position 99) and a glycine residue (at position 194) mutated in this study were distant from the active site, but the degrading activity was strongly affected by their mutation.

• The Journal of Asian Finance, Economics and Business
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• v.7 no.8
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• pp.419-424
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• 2020

This study analyzes the new GMCF method applied by the company with the aim to find out how the production of Accounting Information Systems (AIS) implemented by the company can be managed properly. The study also seeks to find out whether the company needs new system support facilities to facilitate the production performance reporting process of each division and evaluate the performance of GMCF systems in the company. The methods used are descriptive analysis techniques and statistical tests of Paired Sample T-Test comparison; this study uses production data of each unit of a product with random sampling to determine the level of product damage and compare production with the GMCF system and prior to using it. The results of the analysis found that the application of goods mutation control forms (GMCF) greatly influenced the smooth production reporting process, which resulted in an increase in achieving production targets and reducing the risk of product damage during the production process. The company also benefits from the efficiency of production costs when using the GMCF system and can quickly design policies for products that are damaged during the production process. In addition, the company can have damaged products repaired faster than before.

• Journal of Electrical Engineering and Technology
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• v.13 no.3
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• pp.1202-1211
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• 2018

In this paper, induction machine operation efficiency and torque is improved using Machine Learning based Gene Optimization (ML-GO) Technique is introduced. Optimized Genetic Algorithm (OGA) is used to select the optimal induction machine data. In OGA, selection, crossover and mutation process is carried out to find the optimal electrical machine data for induction machine design. Initially, many number of induction machine data are given as input for OGA. Then, fitness value is calculated for all induction machine data to find whether the criterion is satisfied or not through fitness function (i.e., objective function such as starting to full load torque ratio, rotor current, power factor and maximum flux density of stator and rotor teeth). When the criterion is not satisfied, annealed selection approach in OGA is used to move the selection criteria from exploration to exploitation to attain the optimal solution (i.e., efficient machine data). After the selection process, two point crossovers is carried out to select two crossover points within a chromosomes (i.e., design variables) and then swaps two parent's chromosomes for producing two new offspring. Finally, Adaptive Levy Mutation is used in OGA to select any value in random manner and gets mutated to obtain the optimal value. This process gets iterated till finding the optimal value for induction machine design. Experimental evaluation of ML-GO technique is carried out with performance metrics such as torque, rotor current, induction machine operation efficiency and rotor power factor compared to the state-of-the-art works.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.26 no.4
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• pp.318-323
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• 1981

This study was conducted to establish an effective radiation treatment and selection method for induced mutants in M_1 population of soybean treated with gamma-ray. About 64% of total M_1 plants was reduced in plant height up to 50 - 60% and among which 60 - 70% of the plants were contained mutations in M_2 generation. About 60% of the MI plants have born 6 - 15 seeds per plant and 50 - 60% of their progenies produced mutants in M_2 generation. Positive correlation between plant height and number of seeds per plant in M_1 population was found. Higher visible macro-mutation rate in M_2 was observed in the groups of reduced plant height and seed number in the M_1 generation, whereas the frequency of chlorophyll mutation was increased in the group of less damaged plants. The size of mutation sector was increased with reduction in number of seeds per M_1 plant and the mutants were occurred at random in all the parts of M_1 plants. For the effective selection of mutants in soybean mutation breeding, the M_1 seeds should be harvested from the radiation damaged M_1 plants with the application of higher doses of mutagens, and handling M_2 generation by bulk population method is recommendable.