• 정보처리학회논문지C
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• 제16C권5호
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• pp.563-574
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• 2009

랜덤대치 기법은 실용적인 프라이버시 보호 방법으로 다양한 응용 가능성과 프라이버시 손상 관점의 안전성을 보장할 수 있다는 장점이 있다. 하지만 데이터 유용성을 위한 랜덤대치 기법의 정확성을 향상시키는 방법에 대해서는 그동안 면밀히 연구되지 않았다. 본 논문에서는 랜덤 대치 기법의 표준오차에 대한 보다 진전된 이론적 분석을 실시함으로써 정확성을 개선할 수 있는 알고리즘을 제안한다. 다양한 실험을 통하여 균등분포와 정규분포를 따르는 원본 데이터에 대한 랜덤대치 기법의 적용이 실용적이지 못한 정확성을 나타낸다는 사실과 함께 개선된 알고리즘의 정확성 향상 정도를 확인한다. 우리가 제안하는 알고리즘은 기존의 랜덤대치 기법과 동일한 프라이버시 수준을 유지한 상태에서 정확성을 원하는 수준만큼 높일 수 있는 방법이며, 이를 위해 추가로 소요되는 계산량은 실용적인 면에서 여전히 수용 가능한 것임을 밝힌다.

• 한국정보처리학회:학술대회논문집
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• 한국정보처리학회 2007년도 추계학술발표대회
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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.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2000년도 Proceedings of 2000 KSAM International Symposium and Spring Meeting
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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.

• 한국정보처리학회:학술대회논문집
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• 한국정보처리학회 2009년도 춘계학술발표대회
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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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• 제57권1호
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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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• 제35권3호
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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.

• 한국잡초학회지
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• 제18권1호
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• pp.76-83
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• 1998

피속 잡초 수집종 33종을 대상으로 RAPD marker를 이용하여 피 수집종 간의 유전적변이를 알아보고, 수집종들을 판별할 수 있는 DNA marker를 선발하기 위하여 본 실험을 수행한 결과를 요약하면 다음과 같다. Operon사에서 제작된 74개의 10-mer RAPD primer 가운데에서 명확한 다형성을 보이는 6개 primer를 선발하였다. 이들 primer로 PCR에서 증폭된 밴드는 31개이었으며 이 가운데 다형성을 나타내는 band는 26개(83.9%)로 나타났다. 피 수집종 간의 유연 관계를 분석한 결과 공시된 피 수집종은 크게 3그룹으로 분류할 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제11권1호
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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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• 제22권5호
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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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• 제54권2호
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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.