• The KIPS Transactions:PartC
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• v.16C no.5
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• pp.563-574
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• 2009

The merits of random substitutions are various applicability and security guarantee on the view point of privacy breach. However there is no research to improve the accuracy of random substitutions. In this paper we propose an algorithm for improving the accuracy of random substitutions by an advanced theoretical analysis about the standard errors. We examine that random substitutions have an unpractical accuracy level and our improved algorithm meets the theoretical results by some experiments for data sets having uniform and normal distributions. By our proposed algorithm, it is possible to upgrade the accuracy level under the same security level as the original method. The additional cost of computation for our algorithm is still acceptable and practical.

• Proceedings of the Korea Information Processing Society Conference
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• 2007.11a
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• pp.1131-1134
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• 2007

본 논문에서는 랜덤대치(random substitution) 기법에 대하여 심도 있는 분석을 실시한다. 랜덤대치 기법의 효율적인 구현을 위하여 데이터 재구축(reconstruction) 과정에서 필요로 하는 역행렬을 구하는 공식을 제시한다. 또한, 랜덤대치에 사용되는 다양한 파라미터들의 의미를 실험적으로 밝혀내며, 정확도와 프라이버시를 합리적으로 측정할 수 있는 새로운 측도(measure)들을 제안한다.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2000.04a
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• pp.168-171
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• 2000

The thermal stability and catalytic activity of phospholipase A$_1$ from Serratia sp. MK1 were improved by an evolutionary molecular engineering. Two thermostable mutants were isolated after sequential rounds of error-prone PCR to introduce random mutations and filter-based screening of the resultant mutant library, and identified as having six (mutant TA3) and seven (mutant TA13) amino acid substitutions, respectively. Different types of the substitutions were found in two mutants, resulting in the increase of nonploar residues (mutant TA3) or changes between side chains within polar or charged residues (mutant TA13). The wild-type and mutant enzymes were purified, and the effect of temperature on their stability and catalytic activity was investigated. The T$\sub$m/ values of TA3 and TA13 were increased by 7 and 11$^{\circ}C$, respectively. Thus, evolutionary molecular engineering was found to be an effective and efficient approach to increasing thermostability without compromising enzyme activity.

• Proceedings of the Korea Information Processing Society Conference
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• 2009.04a
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• pp.1377-1380
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• 2009

랜덤대치 기법은 프라이버시 손상 관점에서 높은 프라이버시를 보존하면서 원본 데이터의 분포를 재구축하여 데이터 유용성을 확보한다. 데이터 유용성을 위한 랜덤대치 기법의 정확성을 높이는 문제는 그동안 면밀히 연구되지 않았다. 본 논문에서는 랜덤대치 기법이 대부분의 데이터에 대해서 상대적으로 낮은 정확성을 보임을 실험을 통해 밝히고, 이론적인 분석과 실험을 바탕으로 정확성을 높일 수 있는 실용적인 알고리즘 개선 방법을 제안한다.

• 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.

• Journal of Microbiology
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• v.35 no.3
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• pp.228-233
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• 1997

The MTF1 gene of Saccharomyces cerevisiae encodes a 43 kDa MITOCHONDRIAL RNA polymerase specificity factor which recognizes mitochondrial promoters to initiate correct transcription. To better understand structure-function of the MTF1 gene as well as the transcription mechanism of mitochondrial RNA polymerase, two cold-sensitive alleles of the MTF1 mutation were isolated by plasmid shuffling method after PCR-based random mutagenesis of the MTF1 gene. The mutation sites were analyzed by nucleotide sequencing. These cs phenotype mtf1 mutants were respiration competent on the nonfermentible glycerol medium at the permissive temperature, but incompetent at 13.deg.C. The cs phenotype allele of the MTF1, yJH147, encoded an L146P replacement. The other cs allele, yJH148, contained K179E and K214M double replacements. Mutations in both alleles were in a region of Mtflp which is located between domains with amino acid sequence similarities to conserved regions 2 and 3 of bacterial s factors.

• Korean Journal of Weed Science
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• v.18 no.1
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• pp.76-83
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• 1998

Echinochloa species maintained by selling for more than 10 years were classified using random amplified polymorphic DNAs(RAPDs) analysis. Seventy-four decamer of randomly sequence markers were used to classify intraspecific variation irt Echinochloa species. The number of amplification products increased with increasing GC content of the primer in the range between 60% and 70% GC. Single-base substitutions of a primer altered amplification, providing new polymorphisms. The size of amplified DNA was mostly between 0.40kbp and 1.4kbp with the most common bands at 1.1kbp. Echinochloa species were detected with 6 primers which generated 26 polymorphic amplified DNAs. By hierarchical cluster analysis, Echinochloa species collected in Korea were divided into three groups. These results revealed that RAPD markers are useful tools for the determination of genetic variations in Echinochloa species.

• Journal of Microbiology and Biotechnology
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• v.11 no.1
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• pp.29-34
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• 2001

The mutant enzymes of N-carbamyl-D-amino aicd amidohydrolase (N-carbamylase) from Agrobacterium radiobacter NRRL B11291, showing a negligible activity, were selected from the library generated by random mutagenesis. From the sequence analysis, these mutants were found to contain the amino acids substitutions at Cys172, Glu47, and Glu146. Previously, Cys172 was reported to be necessary for the enzyme catalysis. The chemical modification of the N-carbamylase by carboxyl group specific chemical reagent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC), resulted in a loss of activity. The replacement of glutamic acids with glutamines by site-directed mutagenesis led to aggregation of the enzymes. Mutant enzymes fused with maltose binding protein (MBP) were expressed in soluble form, but were inactive. These results indicate that two glutamic acid residues play an important role in structure and function of the N-carbamylase. Multiple sequence alignment of the related enzymes revealed that Glu47 and Glu146 are rigidly conserved, which suggests that tese residues are crucial for the structure and function of the functionally related C-N hydrolases.

• Journal of Microbiology and Biotechnology
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• v.22 no.5
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• pp.607-613
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• 2012

Improvement of endoglucanase activity was accomplished by utilizing error-prone rolling circle amplification, supplemented with 1.7 mM $MnCl_2$. This procedure generated random mutations in the Bacillus amyloliquefaciens endoglucanase gene with a frequency of 10 mutations per kilobase. Six mutated endoglucanase genes, recovered from six colonies, possessed endoglucanase activity between 2.50- and 3.12-folds higher than wild type. We sequenced these mutants, and the different mutated sites of nucleotides were identified. The mutated endoglucanase sequences had five mutated amino acids: A15T, P24A, P26Q, G27A, and E289V. Among these five substitutions, E289V was determined to be responsible for the improved enzyme activity. This observation was confirmed with site-directed mutagenesis; the introduction of only one mutation (E289V) in the wild-type endoglucanase gene resulted in a 7.93-fold (5.55 U/mg protein) increase in its enzymatic activity compared with that (0.7 U/mg protein) of wild type.

• BMB Reports
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• v.54 no.2
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• pp.98-105
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• 2021

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a family of DNA sequences originally discovered as a type of acquired immunity in prokaryotes such as bacteria and archaea. In many CRISPR systems, the functional ribonucleoproteins (RNPs) are composed of CRISPR protein and guide RNAs. They selectively bind and cleave specific target DNAs or RNAs, based on sequences complementary to the guide RNA. The specific targeted cleavage of the nucleic acids by CRISPR has been broadly utilized in genome editing methods. In the process of genome editing of eukaryotic cells, CRISPR-mediated DNA double-strand breaks (DSB) at specific genomic loci activate the endogenous DNA repair systems and induce mutations at the target sites with high efficiencies. Two of the major endogenous DNA repair machineries are non-homologous end joining (NHEJ) and homology-directed repair (HDR). In case of DSB, the two repair pathways operate in competition, resulting in several possible outcomes including deletions, insertions, and substitutions. Due to the inherent stochasticity of DSB-based genome editing methods, it was difficult to achieve defined single-base changes without unanticipated random mutation patterns. In order to overcome the heterogeneity in DSB-mediated genome editing, novel methods have been developed to incorporate precise single-base level changes without inducing DSB. The approaches utilized catalytically compromised CRISPR in conjunction with base-modifying enzymes and DNA polymerases, to accomplish highly efficient and precise genome editing of single and multiple bases. In this review, we introduce some of the advances in single-base level CRISPR genome editing methods and their applications.