• Journal of Microbiology and Biotechnology
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• v.27 no.9
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• pp.1609-1616
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• 2017

The complex roles of cell surface modification in the biofilm formation of Campylobacter jejuni, a major cause of worldwide foodborne diarrheal disease, are poorly understood. In a screen of mutants from random transposon mutagenesis, an insertional mutation in the eptC gene (cj0256) resulted in a significant decrease in C. jejuni NCTC11168 biofilm formation (<20%) on major food contact surfaces, such as polystyrene and borosilicate glass, when compared with wild-type cells (p < 0.05). In C. jejuni strain 81-176, the protein encoded by eptC modified cell surface structures, such as lipid A, the inner core of lipooligosaccharide, and the flagellar rod protein (FlgG), by attaching phosphoethanolamine. To assess the role of eptC in C. jejuni NCTC11168, adherence and motility tests were performed. In adhesion assays with glass surfaces, the eptC mutant exhibited a $0.77log\;CFU/cm^2$ decrease in adherence compared with wild-type cells during the initial 2 h of the assay (p < 0.05). These results support the hypothesis that the modification of cell surface structures by eptC affects the initial adherence in biofilm formation of C. jejuni NCTC11168. In motility tests, the eptC mutant demonstrated reduced motility when compared with wild-type cells, but wild-type cells with the transposon inserted in a gene irrelevant to biofilm formation (cj1111c) also exhibited decreased motility to a similar extent as the eptC mutant. This suggests that although eptC affects motility, it does not significantly affect biofilm formation. This study demonstrates that eptC is essential for initial adherence, and plays a significant role in the biofilm formation of C. jejuni NCTC11168.

• 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.

• Journal of the Korea Institute of Information and Communication Engineering
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• v.10 no.12
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• pp.2329-2334
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• 2006

In this paper, we proposed a method complementing failure of combining DNA fragments, defect of conventional contig assembly programs. In the proposed method, very long DNA sequence data are made into a prototype of fragment of about 700 bases that can be analyzed by automatic sequence analyzer at one time, and then matching ratio is calculated by comparing a standard prototype with 3 fragmented clones of about 700 bases generated by the PCR method. In this process, the time for calculation of matching ratio is reduced by Compute Agreement algorithm. Two candidates of combined fragments of every prototype are extracted by the degree of overlapping of calculated fragment pairs, and then degree of combination is decided using a fuzzy reasoning method that utilizes the matching ratios of each extracted fragment, and A, C, G, T membership degrees of each DNA sequence, and previous frequencies of each A, C, G, T. In this paper. DNA sequence combination is completed by the iteration of the process to combine decided optimal test fragments until no fragment remains. For the experiments, fragments or about 700 bases were generated from each sequence of 10,000 bases and 100,000 bases extracted from 'PCC6803', complete protein genome. From the experiments by applying random notations on these fragments, we could see that the proposed method was faster than FAP program, and combination failure, defect of conventional contig assembly programs, did not occur.

• Journal of Microbiology and Biotechnology
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• v.28 no.11
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• pp.1916-1927
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• 2018

Corynebacterium glutamicum is an excellent platform for the production of amino acids, and is widely used in the fermentation industry. Most industrial strains are traditionally obtained by repeated processes of random mutation and selection, but the genotype of these strains is often unclear owing to the absence of genomic information. As such, it is difficult to improve the growth and amino acid production of these strains via metabolic engineering. In this study, we generated a complete genome map of an industrial L-valine-producing strain, C. glutamicum XV. In order to establish the relationship between genotypes and physiological characteristics, a comparative genomic analysis was performed to explore the core genome, structural variations, and gene mutations referring to an industrial L-leucine-producing strain, C. glutamicum CP, and the widely used C. glutamicum ATCC 13032. The results indicate that a 36,349 bp repeat sequence in the CP genome contained an additional copy each of lrp and brnFE genes, which benefited the export of L-leucine. However, in XV, the kgd and panB genes were disrupted by nucleotide insertion, which increase the availability of precursors to synthesize L-valine. Moreover, the specific amino acid substitutions in key enzymes increased their activities. Additionally, a novel strategy is proposed to remodel central carbon metabolism and reduce pyruvate consumption without having a negative impact on cell growth by introducing the CP-derived mutant $H^+$/citrate symporter. These results further our understanding regarding the metabolic networks in these strains and help to elucidate the influence of different genotypes on these processes.

• Korean Journal of Microbiology
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• v.39 no.2
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• pp.76-82
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• 2003

To prepare evolved PHB depolymerase with increased activity for PHB or P(3HB-co-3HV) compared to the activity of the original PHB depolymerase from Alcaligenes faecalis T1, random mutation of the cloned PHB depolymerase gene was performed by using a DNA shuffling method. A library of mutated PHB depolymerase genes from A. faecalis T1 was fused to the ice nucleation protein (INP) gene from Pseudomonas syringae in pJHCl 1 and approximately 7,000 transformants were isolated. Using M9 minimal medium containing PHB or P(3HB-co-3HV) as the carbon source, mutants showing alteration in PHB depolymerase activity were selected from the transformants. The PHB depolymease activity of the transformants was confirmed by the formation of halo around colony and the turbidity decrease tests using culture supermatants. The catalytic activity of PHB depolymerase of the best mutant II-4 for PHB or P(3HB-co-13 mol% 3HV) was approximately 1.8-fold and 3.2-fold, respectively, higher than that of the original PHB depolymerase. DNA sequence analysis revealed that three amino acid residues (Ala209Val, Leu258Phe, and Asp263Thr) were substituted in II-4. From the mutational analysis, it was presumed that the substitution of amino acids near catalytic triad to more hydrophobic amino acids enhance the catalytic activity of PHB depolymerase from A. faecalis T1.

• Proceedings of the Microbiological Society of Korea Conference
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• 2002.10a
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• pp.162-172
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• 2002

Schizosaccharomyces pombe is suited for the study of cytokinesis as it divides by forming a septum in the middle of the cell at the end of mitosis. To enhance our understanding of the cytokinesis, we have carried out a genetic screen for temperature-sensitive S. pombe mutants that show defects in septum formation and cell division. Here we present the isolation and characterization of a new temperature-sensitive mutant, sun1(septum uncontrolled), which undergoes uncontrolled septation during cell division cycle at restrictive temperature $(37^{\circ}C)$. In sun1 mutant, actin ring and septum are positioned at random locations and angles, and nuclear division cycle continues. These observations suggest that the sun] gene product is required for the proper placement of the actin ring as well as precise septation. The sun] mutant is monogenic recessive mutation unlinked to previously known various cdc genes of S. pombe. In a screen for $sunl^+$ gene to complement the sun] mutant, we have cloned a gene, $susl^+$(suppressor of sun1 mutant), that encodes a protein of 689 amino acids. The predicted amino acid sequence of $susl^+$ gene is similar to the human hMadlp and Saccharomyces cerevisiae Mad1p, a component of the spindle checkpoint in eukaryotic cells. The null mutant of $susl^+$ gene grows normally at various temperatures and has the increased sensitivity to anti-microtubule drug, while $susl^+$ mutant shows no sensitivity to microtubule destabilizing drugs. The putative S. pombe Sus1p directly interacts with S. pombe Mad2p in yeast two-hybrid assays. These data suggest that the newly isolated susr gene encodes S. pombe Mad1p and suppresses sun] mutant defective in controlled septation in a cell division cycle.

• Proceedings of the Korean Society for Noise and Vibration Engineering Conference
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• 2006.05a
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• pp.164-170
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• 2006

This paper proposes a RSM-based hybrid evolutionary algorithm (RHEA) which combines the merits of the popular programs such as genetic algorithm (GA), tabu search method, response surface methodology (RSM). This algorithm, for improving the convergent speed that is thought to be the demerit of genetic algorithm, uses response surface methodology and simplex method. The mutation of GA offers random variety to finding the optimum solution. In this study, however, systematic variety can be secured through the use of tabu list. Efficiency of this method has been proven by applying traditional test functions and comparing the results to GA. And it was also proved that the newly suggested algorithm is very effective to find the global optimum solution to minimize the weight for avoiding the resonance of fresh water tank that is placed in the rear of ship. According to the study, GA's convergent speed in initial stages is improved by using RSM method. An optimized solution is calculated without the evaluation of additional actual objective function. In a summary, it is concluded that RHEA is a very powerful global optimization algorithm from the view point of convergent speed and global search ability.

• Transactions of the Korean Society for Noise and Vibration Engineering
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• v.16 no.6 s.111
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• pp.665-673
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• 2006

This paper proposes a RSM-based hybrid evolutionary Algorithm (RHEA) which combines the merits of the popular programs such as genetic algorithm (GA), tabu search method and response surface methodology (RSM). This algorithm, for improving the convergent speed that is thought to be the demerit of genetic algorithm, uses response surface methodology and simplex method. The mutation of GA offers random variety to finding the optimum solution. In this study, however, systematic variety can be secured through the use of tabu list. Efficiency of this method has been proven by applying traditional left functions and comparing the results to GA. It was also proved that the newly suggested algorithm is very effective to find the global optimum solution to minimize the weight for avoiding the resonance of fresh water tank that is placed in the after body area of ship. According to the study, GA's convergent speed in initial stages is improved by using RSM method. An optimized solution is calculated without the evaluation of additional actual objective function. In a summary, it is concluded that RHEA is a very powerful global optimization algorithm from the view point of convergent speed and global search ability.

• Journal of Animal Science and Technology
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• v.51 no.5
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• pp.353-360
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• 2009

When 14 microsatellite (MS) markers were applied in the identifying test for 480 Hanwoo, the discriminating power was estimated as $3.43{\times}10^{-27}$ based on the assumption of a random mating group (PI). This rate is 1,000 times higher than that of 60 single nucleotide polymorphism (SNP) markers. On the other hand, the power of the 60 SNP markers was estimated as $4.69{\times}10^{-20}$ and $8.02{\times}10^{-12}$ on the assumption of a half-sib mating group ($PI_{half-sibs}$) and a full-sib mating group ($PI_{sibs}$), respectively. These powers were 10 times and 10,000 times higher than those of the 14 MS markers. The results indicated that the total number of alleles (MS vs SNP = 146 vs 120) acted as a key factor for the discriminating power in a random mating population, and the total number of markers (MS vs SNP = 14 vs 60) was a dominant influence on the power in half-sib and full-sib populations. In the Hanwoo population, in which it was assumed that the entire population is the enormous half-sib group formed by the absolute genetic contribution of a few nuclear bulls, there will be only a 10 times difference in the discriminating power between the 14 MS markers and the 60 SNP makers. However, the probability of not excluding a candidate parent pair from the parentage of an arbitrary offspring, given that only the genotype of the offspring ($PNE_{pp}$) was 1,000 times higher as shown by the 14 MS markers than that by the 60 SNP markers. The strong points of SNP makers are the stability of the variation (low mutation rate) and automation of high-throughput genotyping. In order to apply these merits for the practical and constant Hanwoo identity test, research and development are required to set a cost-effective platform and produce a homemade apparatus for SNP genotyping.

• Childhood Kidney Diseases
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• v.16 no.2
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• pp.73-79
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• 2012

Purpose: The association of mitochondrial DNA (mtDNA) mutations, deletions and copy number with progressive changes in patients with some glomerular disease and end-stage renal disease have been reported. In this study, we performed mtDNA mutation analysis in children with IgA nephropathy to investigate its role in progressive clinical course. Methods: Seven children with IgA nephropathy were involved in this study. MtDNA isolated from platelet was amplified by PCR and sequenced entirely. Results: The mean age at renal biopsy was $11.5{\pm}2.2$ year and the mean age at latest evaluation was $17.9{\pm}3.2$ year. The mean follow-up period were $7.8{\pm}3.1$ years. Patients was divided into 2 groups according to the amount of proteinuria at presenting manifestation. Group 2 patients were nephrotic syndrome. Renal function reveals within normal range in all patients. In group 2 patients, the mean serum albumin level was significantly lower than those of group 1 ($3.7{\pm}0.6g/dL$ vs. $4.7{\pm}0.2g/dL$, P=0.0241) and the mean total cholesterol level was significantly higher than those of group 1 ($222.7{\pm}35.7mg/dL$ vs. $148.3{\pm}29.1mg/dL$, P=0.0283). In Group 2 patients, total amount of protein of 24 hour collected urine also significantly higher than those of group 1 ($1,466.0{\pm}742.5mg$ vs. $122.5{\pm}48.1mg$, P=0.0135). Pr/Cr ratio in random urine sample was also higher in group 2 than those of group 1 but the statistical significance was not noted ($1.8{\pm}1.6$ vs. $0.2{\pm}0.2$, P=0.0961). Deletion of mtDNA nt 8272-8281 were observed in two patients, one patient in each groups, respectively. This is noncoding lesion. No patients demonstrated the mtDNA mutations. Conclusions: We have identified a deletion of mtDNA nt 8272-8281 in two children with IgA nephropathy. Further studies are needed to clarify the role of mitochondrial function in the progressive change of IgA nephropathy.