• Animal cells and systems
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• v.8 no.3
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• pp.205-212
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• 2004

The tumor suppressor protein p53 is stabilized by the herpes-virus-associated ubiquitin-specific protease (HAUSP), a deubiquitinating enzyme. We previously isolated and characterized a mouse orthologue of HAUSP, mHAUSP. mHAUSP cDNA consisted of 3,312 bp encodes 1,103 amino acids with a molecular weight of approximately 135 kDa containing highly conserved Cys, Asp (I), His, and Asn/Asp (II) domains. In this study, we carried out site-directed mutagenesis of 6 conserved amino acids (Cys224, Gln231, Asp296, His457, His465, and Asp482) in Cys box, QQD box, and His box. Interestingly, the conserved Gln 231 was not essential for the catalytic activity of mHAUSP. However, the other conserved amino acids were required for deubiquitinating activity of mHAUSP. We performed isopeptidase assay and confirmed that mHAUSP is able to remove ubiquitin from ubiquitinated substrates. In addition, we observed that mHAUSP induces apoptosis in HeLa cells.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.144.1-144
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• 2003

Attenuated viruses can protect their hosts against challenge to their related viruses. Increasing evidence shows that mutations of the tobamoviral 126/183 kDa protein play a major role in the viral attenuation and contribute to the cross protection mechanism. In this study, four mutants of Pepper mild mottle virus (PMMoV) have been constructed by mutagenesis; two mutants, pTPpoly348 and pTPpoly762, were substituted in the middle of replicase gene, and the others, pTPL3D:: $\Delta$6207 and pTPL3D:: $\Delta$6219, were deletion mutants made by deleting some parts of pseudoknot structures of the 3' noncoding region (NCR) of the virus. Progeny viruses generated from the four mutants were infectious on N. benthamiana plants with symptomless or mild mosaic symptom. Replication efficiency and viral product accumulations of four mutants were assessed by Northern and Western blot analyses on BY-2 protoplast cells. Accumulation of CP for the pTPL3D:: $\Delta$6207 and pTPL3D:: $\Delta$6219 were lower than that of other mutants and wild type virus. These data suggest that the 3'-NCR mutations contribute to the viral gene expression in host tissues, while mutants of replicase gene rather govern the symptom expression.

• BMB Reports
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• v.42 no.12
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• pp.788-793
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• 2009

TLX2 is an orphan homeodomain transcription factor whose expression is mainly associated with tissues derived from neural crest cells. Recently, we have demonstrated that PHOX2A and PHOX2B are able to enhance the neural cell-type specific expression of human TLX2 by binding distally the 5' -flanking region. In the present work, to deepen into the TLX2 transcription regulation, we have focused on the proximal 5'-flanking region of the gene, mapping the transcription start site and identifying a minimal promoter necessary and sufficient for the basal transcription in cell lines from different origin. Site-directed mutagenesis has allowed to demonstrate that the integrity of this sequence is crucial for gene expression, while electrophoretic mobility shift assays and chromatin immunoprecipitation experiments have revealed that such an activity is dependent on the binding of a PBX factor. Consistent with these findings, such a basal promoter activity has resulted to be enhanced by the previously reported PHOX2-responding sequence.

• The Plant Pathology Journal
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• v.28 no.4
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• pp.373-380
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• 2012

Burkholderia pyrrocinia CH-67 is a biocontrol bacterium with strong antifungal activity against several plant pathogenic fungi. Transposon mutagenesis was performed to identify the genes responsible for the antifungal activity of B. pyrrocinia CH-67. Of the 2,500 mutants tested using the Fulvia fulva spore screening method, a mutant deficient in antifungal activity, M208, was selected. DNA sequence analysis of the transposon-inserted region revealed that a gene encoding an adenylate kinase-related kinase was disrupted in M208. Antifungal activity was restored in M208 when a full-length adenylate kinase gene with its promoter was introduced in trans. The deduced amino acid sequence of adenylate kinase from CH-67 was 80% identical to that of B. cenocepacia MCO-3. Adenosine diphosphate supplementation or high levels of adenosine triphosphate and adenosine monophosphate together restored antifungal activity in M208, suggesting that adenylate kinase of B. pyrrocinia CH-67 is involved in antifungal activity expression.

• Proceedings of the Botanical Society of Korea Conference
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• 1985.08b
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• pp.59-71
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• 1985

In rice, limited efforts have been made to identify genes by the use of insertional mutagens, especially heterologous transposons such as the maize Ac/Ds. We constructed Ac and gene trap Ds vectors and introduced them into the rice genome by Agrobacterium-mediated transformation. In this report, rice plants that contained single and simple insertions of T-DNA were analyzed in order to evaluate the gene-tagging efficiency. The 3'end of Ds was examined for putative splicing donor sites. As observed in maize, three splice donor sites were identified at the 3'end of the Ds in rice. Nearly 80% of Ds elements wered excised from the original T-DNA sites, when Ac cDNA was expressed under a CaMV 35S promoter. Repetitive ratoon culturing was performed to induce new transpositions of Ds in new plants derived from cuttings. About 30% of the plants carried at least one Ds that underwent secondary transposition in the later cultures. 8% of transposed Ds elements expressed GUS in various tissues of rice panicles. With cloned DNA adjacent to Ds, the genomic complexities of the insertion sites were examined by Southern hybridization. Half of the Ds insertion sites showed simple hybriodization patterns which could be easily utilized to locate the Ds. Our data demonstrate that the Ac/Ds mediated gene trap system could prove an excellent tool for the analysis of functions of genes in rice. We discuss genetic strategies that could be employed in a largee scale mutagenesis using a heterologous Ac/Ds family in rice.

• Korean Journal of Microbiology
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• v.22 no.4
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• pp.265-269
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• 1984

For the utilization as donor cells of conjugation, DAP-Auxotrophs were isolated from C. coli cells, carrying plasmid $p^{RD1}$ with(a) drug resistance makers from Pseudononas $(Km^r, \;Carb^r, \;Tc^r)$ and (b) the nif-gene group from Klebsiela. E. coli $p^{RD1}$ cells were treated with nitrosoguanidine for the mutagenesis and cephalexin for the isolation of DAP-Auxotrophs. The nature of auxotrophs was verified by suitable biochemical test and checking with 6-cyanopurine as a color indicator for the presence of nif-gene.

• The Plant Pathology Journal
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• v.37 no.4
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• pp.389-395
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• 2021

Soil is the major source of plant-associated microbes. Several fungal and bacterial species live within plant tissues. Actinomycetes are well known for producing a variety of antibiotics, and they contribute to improving plant health. In our previous report, Streptomyces globisporus SP6C4 colonized plant tissues and was able to move to other tissues from the initially colonized ones. This strain has excellent antifungal and antibacterial activities and provides a suppressive effect upon various plant diseases. Here, we report the genome-wide analysis of antibiotic producing genes in S. globisporus SP6C4. A total of 15 secondary metabolite biosynthetic gene clusters were predicted using antiSMASH. We used the CRISPR/Cas9 mutagenesis system, and each biosynthetic gene was predicted via protein basic local alignment search tool (BLAST) and rapid annotation using subsystems technology (RAST) server. Three gene clusters were shown to exhibit antifungal or antibacterial activity, viz. cluster 16 (lasso peptide), cluster 17 (thiopeptide-lantipeptide), and cluster 20 (lantipeptide). The results of the current study showed that SP6C4 has a variety of antimicrobial activities, and this strain is beneficial in agriculture.

• Proceedings of the Korean Society of Crop Science Conference
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• 2022.10a
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• pp.24-24
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• 2022

This present study was to identify a novel candidate gene that contribute to the elevated α-linolenic acid (ALA, ω-3) concentration in PE2166 from mutagenesis of Pungsannamul. Major loci qALA5_1 and qALA5_2 were detected on chromosome 5 of soybean through quantitative trait loci mapping analyses of recombinant inbred lines. With next generation sequencing of parental lines and Pungsannamul, and recombinant analyses, a potential gene, Glyma. 05g221500 (HD) controlling elevated ALA concentration was identified. HD is a homeodomain-like transcriptional regulator that may regulate the expression level of microsomal ω-3 fatty acid desaturase (FAD3) genes responsible for the conversion of linoleic acid into ALA in the fatty acid biosynthetic pathway. In addition, we hypothesized that combination of mutant alleles, HD and either of microsomal delta-12 fatty acid desaturase 2-1 (FAD2-1\ could reduce the ω-6/ω-3 ratio. In populations where HD, and FAD2-1A and FAD2-1B genes were segregated, combination of a hd allele from PE2166 and either of the variant FAD2-1 alleles were sufficient to reduce the ω-6/ω-3 ratio in seeds.

• Journal of Life Science
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• v.23 no.3
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• pp.347-354
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• 2013

Innate immunity is the ability to differentiate infectious agents from self. The innate immune system is comprised of a complicated network of recognition and effector molecules that act together to protect the host in the early stage of an infectious challenge. Mannose-binding lectin (MBL or mannose-binding protein, MBP) belongs to the family of $Ca^{2+}$-dependent lectins (C-type lectin with a collagen-like domain), which are considered an important component of innate immunity. While it is associated with increased risk and severity of infections and autoimmunity, the most frequent immuno-deficiency syndrome was reported to be low MBL level in blood. Deficiency of human MBL is caused by mutations in the coding region of the MBL gene. Rat homologue gene of human MBL gene was used to study functions of wild type and mutant MBL proteins. Although extensive studies have yielded the structural information of MBL, the functions of MBL, especially mutant MBL, still require investigation. We previously reported the cloning of rat wild-type MBL gene and the production of a truncated form of MBL protein and its antibody. Here, we present the cloning of mutant MBL cDNA in collagen-like domain (R40C, G42D, and G45E) using site-directed mutagenesis and differential behaviors of wild type and mutant MBL in cells. The major difference between wild type and mutant MBL was that while wild type MBL was secreted, mutant MBL was inhibited for secretion, retained in endoplasmic reticulum, and still functioned as a lectin.

• Journal of Life Science
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• v.6 no.3
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• pp.185-192
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• 1996

Bacteriophage T7 gene 2.5 protein, a single-stranded DNA binding protein, is required for T7 DNA replication, recombination, and repair. T7 gene 2.5 protein has two distinctive domains, DNA binding and C-terminal domain, directly involved in protein-protein interaction. Gene 2.5 protein participates in the DNA replication of Bacteriophage T7, which makes this protein essential for the T7 growth and DNA replication. What gene 2.5 protein makes important at T7 growth and DNA replication is its binding affinity to single-stranded DNA and the protein-protein important at T7 DNA replication proteins which are essential for the T7 DNA synthesis. We have constructed pGST2.5(WT) encoding the wild-type gene 2.5 protein and pGST2.5$\Delta $21C lacking C-terminal 21 amino acid residues. The purified GST-fusion proteins, GST2.5(WT) and GST2.5(WT)$\Delta$21C, were used for whether the carboxyl-terminal domain participates in the protein-protein interactions or not. GST2.5(WT) and GST2.5$\Delta$21C showed the difference in the protein-protein interaction. GST2.5(WT) interacted with T7 DNA polymerase and gene 4 protein, but GST2.5$\Delta$21C did not interact with either protein. Secondly, GST2.5(WT) interacts with gene 4 proteins (helicase/primase) but not GST2.5$\Delta$21C. these results proved the involvement of the carboxyl-terminal domain of gene 2.5 protein in the protein-protein interaction. We clearly conclude that carboxy-terminal domain of gene 2.5 protein is firmly involved in protein-protein interactions in T7 replication proteins.