• KSBB Journal
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• v.25 no.2
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• pp.167-172
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• 2010

Streptomyces scabiei producing phytotoxin called thaxtomin, which cause scab disease on economically important crops such as potato. For molecular genetics study of S. scabiei an effective transformation method was established based on conjugal transfer from Escherichia coli ET12567 (pUZ8002) using a phiC31-derived integration vector, pSET152, containing oriT and attP fragments. The high frequency was obtained on MS medium containing 50 mM $MgCl_2$. In addition, the sequence and location of the chromosomal integration attB site of S. scabiei was identified for the first time in the strains producing thaxtomin by the southern blot analysis of exconjugants and the sequencing of plasmid containing DNA flanking the insertion sites from exconjugant chromosome. Similar to the case of Streptomyces species, a single phiC31 attB site of S. scabiei is present within an ORF encoding a pirin-homolog.

• The Plant Pathology Journal
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• v.26 no.1
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• pp.8-16
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• 2010

The phytopathogenic fungus Magnaporthe oryzae is a major limiting factor in rice production. To understand the genetic basis of M. oryzae pathogenic development, we previously analyzed a library of T-DNA insertional mutants of M. oryzae, and identified ATMT0879A1 as one of the pathogenicity-defective mutants. Molecular analyses and database searches revealed that a single TDNA insertion in ATMT0879A1 resulted in functional interference with an annotated gene, MGG00056, which encodes a short-chain dehydrogenase/reductase (SDR). The mutant and annotated gene were designated as $MoSDR1^{T-DNA}$ and MoSDR1, respectively. Like other SDR family members, MoSDR1 possesses both a cofactor-binding motif and a catalytic site. The expression pattern of MoSDR1 suggests that the gene is associated with pathogenicity and plays an important role in M. oryzae development. To understand the roles of MoSDR1, the deletion mutant ${\Delta}Mosdr1$ for the gene was obtained via homology-dependent gene replacement. As expected, ${\Delta}Mosdr1$ was nonpathogenic; moreover, the mutant displayed pleiotropic defects in conidiation, conidial germination, appressorium formation, penetration, and growth inside host tissues. These results suggest that MoSDR1 functions as a key metabolic enzyme in the regulation of development and pathogenicity in M. oryzae.

• Genomics & Informatics
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• v.3 no.2
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• pp.61-65
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• 2005

Comparing 231 genes on chimpanzee chromosome 22 with their orthologous on human chromosome 21, we have found that 15 orthologs have indels within their coding sequences. It was rather surprising that significant number of genes have changed by indel, despite the shorter time since their divergence and led us hypothesize that indels and structural changes may represent one of the major mechanism of proteome evolution in the higher primates. Human T-complex protein 10 like (TCP 10L) is a representative having indel within its coding sequence. Gene structure of human TCP10L compared with chimpanzee TCP10L gene showed 16 base pair difference in genomic DNA. As a result of the indel, frame shift mutation occurs in coding sequence (CDS) and human TCP10L express longer polypeptide of 21 amino acid residues than that of chimpanzee. Our prediction found that the indel may affect to dramatic change of secondary protein structure between human and chimpanzee TCP10L. Especially, the structural changes in the C-terminal region of TCP10L protein may affect on the interacting potential to other proteins rather than DNA binding function of the protein. Through these changes, TCP10L might influence gene expression profiles in liver and testis and subsequently influence the physiological changes required in primate evolution.

• Journal of Microbiology
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• v.37 no.4
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• pp.248-255
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• 1999

We have analyzed the MRE3/REC114 gene of Saccharomyces cerevisiae, previously detected in isolation of mutants defective in meiotic recombination. We cloned the MRE3/REC114 gene by complementation of the meiotic recombination defect and it has been mapped to chormosome XIII. The DNA sequence analysis revealed that the MRE3 gene is identical to the REC114 gene. The upstream region of the MRE3/REC114 gene contains a T_4C site, a URS (upstream repression sequence) and a TR (T-rich) box-like sequence, which reside upstream of many meiotic genes. Coincidentally, northern blot analysis indicated that the three sizes of MRE3/REC114 transcripts, 3.4, 1.4 and 1.2 kb, are induced in meiosis. A less abundant transcript of 1.4 kb is detected in both mitotic and meiotic cells, suggesting that it is needed in mitosis as well as meiosis. To examine the role of the MRE3/REC114 gene, we constructed mre3 disruption mutants. Strains carrying an insertion or null deletion of the MRE3/REC114 gene showed slow growth in nutrient medium and the doubling time of these cells increased approximately by 2-fond compared to the wild-type strain. Moreover, the deletion mutant (${\delta}$mre3) displayed no meiotically induced recombination and no viable spores. The mre3/rec114 spore lethality can be suppressed by spo13, a mutation that causes cells to bypass reductional division. The double-stranded breaks (DSBs) which are involved in initiation of meiotic recombination were not detected in the analysis of meiotic chromosomal DNA from the mre3/rec114 disruptant. From these results we suggest that the MRE3/REC114 gene product is essential in normal growth and in early meiotic stages involved in meiotic recombination.

• Molecules and Cells
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• v.20 no.3
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• pp.385-391
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• 2005

As a first step towards identifying genes involving in the signal transduction pathways mediating rice blast resistance, we isolated 3 mutants lines that showed enhanced susceptibility to rice blast KJ105 (91-033) from a T-DNA insertion library of the japonica rice cultivar, Hwayeong. Since none of the susceptible phenotypes co-segregated with the T-DNA insertion we adapted a map-based cloning strategy to isolate the gene(s) responsible for the enhanced susceptibility of the Hwayeong mutants. A genetic mapping population was produced by crossing the resistant wild type Hwayeong with the susceptible cultivar, Nagdong. Chi-square analysis of the $F_2$ segregating population indicated that resistance in Hwayeong was controlled by a single major gene that we tentatively named Pi-hy. Randomly selected susceptible plants in the $F_2$ population were used to build an initial map of Pi-hy. The SSLP marker RM2265 on chromosome 2 was closely linked to resistance. High resolution mapping using 105 $F_2$ plants revealed that the resistance gene was tightly linked, or identical, to Pib, a resistance gene with a nucleotide binding sequence and leucine-rich repeats (NB-LRR) previously isolated. Sequence analysis of the Pib locus amplified from three susceptible mutants revealed lesions within this gene, demonstrating that the Pi-hy gene is Pib. The Pib mutations in 1D-22-10-13, 1D-54-16-8, and 1C-143-16-1 were, respectively, a missense mutation in the conserved NB domain 3, a nonsense mutation in the 5th LRR, and a nonsense mutation in the C terminus following the LRRs that causes a small deletion of the C terminus. These findings provide evidence that NB domain 3 and the C terminus are required for full activity of the plant R gene. They also suggest that alterations of the resistance gene can cause major differences in pathogen specificity by affecting interactions with an avirulence factor.

• Korean Journal of Microbiology
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• v.41 no.2
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• pp.140-145
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• 2005

Streptomyces natalensis ATCC27448 produces natamycin, a commercially important macrolide antifungal antibiotic. For molecular genetic study of S. natalensis, we have developed a system for introducing DNA into S. natalensis via conjugal transfer from Escherichia coli. An effective transformation procedure for S. natalensis was established based on transconjugation from E, coli ET12567/pUZ8002 using a ${\Phi}C31$-derived integration vector, pSET152, containing oriT and attP fragments. The high frequency was obtained on MS medium containing 10 mM $MgCl_2$ using $6.25\times10^8$ of E.coli donor cells without heat treatment of spores. In addition, southern blot analysis of exconjugants and the sequence of plasmids containing DNA flanking the insertion sites from the chromosome revealed that S. natalensis contains a single ${\Phi}C31$ attB site and at least a secondary or pseudo attB site. Similar to the case of various Streptomyces species, a single ${\Phi}C31$ attB site of S. natalensis is present within an ORF encoding a pirin-homolog, but a pseudo-attB site is present within a distinct site (GenBank accession no. $YP\_117731$) and also its sequence deviates from the consensus sequences of attB sequence.

• Journal of Periodontal and Implant Science
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• v.52 no.2
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• pp.155-169
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• 2022

Purpose: The aim of this study was to determine the effect of insulin growth factor binding protein-3 (IGFBP-3) on the inhibition of glucose oxidative stress and promotion of bone formation near the implant site in a rat model of methylglyoxal (MGO)-induced bone loss. Methods: An in vitro study was performed in MC3T3 E1 cells treated with chitosan gold nanoparticles (Ch-GNPs) conjugated with IGFBP-3 cDNA followed by MGO. An in vivo study was conducted in a rat model induced by MGO administration after the insertion of a dental implant coated with IGFBP-3. Results: MGO treatment downregulated molecules involved in osteogenic differentiation and bone formation in MC3T3 E1 cells and influenced the bone mineral density and bone volume of the femur and alveolar bone. In contrast, IGFBP-3 inhibited oxidative stress and inflammation and enhanced osteogenesis in MGO-treated MC3T3 E1 cells. In addition, IGFBP-3 promoted bone formation by reducing inflammatory proteins in MGO-administered rats. The application of Ch-GNPs conjugated with IGFBP-3 as a coating of titanium implants enhanced osteogenesis and the osseointegration of dental implants. Conclusions: This study demonstrated that IGFBP-3 could be applied as a therapeutic component in dental implants to promote the osseointegration of dental implants in patients with diabetes, which affects MGO levels.

• Preventive Nutrition and Food Science
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• v.9 no.4
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• pp.324-329
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• 2004

In order to investigate the molecular mechanism of $\gamma$-aminobutyric acid (GABA) production in lactic acid bacteria, we cloned a glutamate decarboxylase (GAD) gene from Lactobacillus plantarum using polymerase chain reaction (PCR). One PCR product DNA was obtained and inserted into a TA cloning vector with a T7 promoter. The recombinant plasmid was used to transform E. coli. The insertion of the product was con­firmed by EcoRI digestion of the plasmid purified from the transformed E. coli. Nucleotide sequence analysis showed that the insert is a full-length Lactobacillus plantarum GAD and that the sequence is $100\%$ and $72\%$ identical to the regions of Lactobacillus plantarum GAD and Lactococcus lactis GAD sequences deposited in GenBank, accession nos: NP786643 and NP267446, respectively. The amino acid sequence deduced from the cloned Lactobacillus plantarum GAD gene showed $100\%$ and $68\%$ identities to the GAD sequences deduced from the genes of the NP786643 and NP267446, respectively. To express the GAD protein in E. coli, an expression vector with the GAD gene (pkk/GAD) was constructed and used to transform the UT481 E. coli strain and the expression was confirmed by analyzing the enzyme activity. The Lactobacillus plantarum GAD gene obtained may facilitate the study of the molecular mechanisms regulating GABA metabolism in lactic acid bacteria.

• International Journal of Stem Cells
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• v.16 no.2
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• pp.234-243
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• 2023

The recent advances in human pluripotent stem cells (hPSCs) enable to precisely edit the desired bases in hPSCs to be used for the establishment of isogenic disease models and autologous ex vivo cell therapy. The knock-in approach based on the homologous directed repair with Cas9 endonuclease, causing DNA double-strand breaks (DSBs), produces not only insertion and deletion (indel) mutations but also deleterious large deletions. On the contrary, due to the lack of Cas9 endonuclease activity, base editors (BEs) such as adenine base editor (ABE) and cytosine base editor (CBE) allow precise base substitution by conjugated deaminase activity, free from DSB formation. Despite the limitation of BEs in transition substitution, precise base editing by BEs with no massive off-targets is suggested to be a prospective alternative in hPSCs for clinical applications. Considering the unique cellular characteristics of hPSCs, a few points should be considered. Herein, we describe an updated and optimized protocol for base editing in hPSCs. We also describe an improved methodology for CBE-based C to T substitutions, which are generally lower than A to G substitutions in hPSCs.

• Journal of Life Science
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• v.28 no.9
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• pp.1030-1041
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• 2018

Abiotic stresses limit the growth and productivity of plants. Cellular adaptation to abiotic stresses requires coordinated regulation in gene expression directed by complex mechanisms. This study used the activation tagging system to identify novel salt stress-responsive genes. The study selected 9 activation tagging lines that showed salt stress-tolerant phenotypes during their germination stages. Thermal asymmetric interlaced-PCR (TAIL-PCR) was used to identify the T-DNA tagging sites on the Arabidopsis genome in selected activation tagging lines, including AT7508, AT7512, AT7527, AT7544, AT7548, and AT7556. RT-PCR analysis showed that ClpC2/HSP93-III (At3g48870), plant thionin family (At2g20605), anti-muellerian hormone type-2 receptor (At3g50685), vacuolar iron transporter family protein (At4g27870), and microtubule-associated protein (At5g16730) were activated in AT7508, AT7512, AT7527, AT7544, and AT7556, respectively. Interestingly, in AT7548, both the genes adjacent to the T-DNA insertion site were activated: Arabinogalactan protein 13 (AGP13) (At4g26320) and F-box/RNI-like/FBD-like domains-containing protein (At4g26340). All of the seven genes were newly identified as salt stress-responsive genes from this study. Among them, the expression of ClpC2/HSP93-III, AGP13, F-box/RNI-like/FBD-like domains-containing protein gene, and microtubule-associated protein gene were increased under salt-stress condition. In addition, AT7508, AT7527, and AT7544 were more tolerant to salt stress than wild type at seedling development stage, functionally validating the screening results of the activation tagging lines. Taken together, our results demonstrate that the activation tagging system is useful for identifying novel stress-responsive genes.