• BMB Reports
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• v.28 no.6
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• pp.484-489
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• 1995

The product of bacteriophage T7 gene 2.5 is a single-stranded DNA binding protein and plays an important role in T7 DNA replication, recombination, and repair. Genetic analysis of T7 phage defective in gene 2.5 shows that the gene 2.5 protein is essential for T7 DNA replication and growth (Kim and Richardson, 1993). The C-terminal truncated gene 2.5 protein ($GP2.5-{\Delta}21C$) cannot substitute for wild-type gene 2.5 protein in vivo; suggesting that the C-terminal domain of gene 2.5 protein is essential for protein-protein interactions (Kim and Richardson, 1994; J. Biol. Chem. 269, 5070-5078). Truncated gene 2.5 proteins lacking 19 residues ($GP2.5-{\Delta}19N$) and 39 residues ($GP2.5-{\Delta}39N$) from the amino-terminal domain were constructed by in vitro mutagenesis. $GP2.5-{\Delta}19N$ can support the growth of T7 phage lacking gene 2.5 while $GP2.5-{\Delta}39N$ cannot substitute for wild-type gene 2.5 protein in vivo; however, its ability to bind to single-stranded DNA is not affected. These results clearly demonstrate that the 20~39 amino-terminal region of gene 2.5 protein is required for T7 growth in vivo but may not be involved in DNA binding activity.

• Korean Journal of Microbiology
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• v.52 no.3
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• pp.298-305
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• 2016

Previously our laboratory showed that Pseudomonas alkylphenolica KL28 possesses two different lap and pcu gene clusters for p-cresol catabolism. In this study, additional gene cluster (pchACXF-pcaHG-orf4-pcaBC) has been identified to encode enzymes necessary for catabolism of p-cresol to ${\beta}$-carboxy-cis,cis-muconate. This gene cluster showed almost identical nucleotide sequence homologies to those in the plasmid of Pseudomonas putida NCIMB 9866 and 9869, British origins, indicating the possibility of a horizontal gene transfer. Through mutagenesis of each gene cluster and gfp-based promoter reporter assays, it has been shown that the three gene clusters are functionally operated and pch genes are induced by p-cresol. Furthermore, the pcu gene cluster of the three was shown to be dominantly expressed in utilization of p-cresol. Mutation of the pcu gene was defective in aerial structure formation under p-cresol vapor, indicating the utilization rate of carbon source is one of key elements for the multicellular development of this strain.

• Plant Breeding and Biotechnology
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• v.6 no.4
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• pp.313-320
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• 2018

Rice is the staple food of more than 50% of the world population. Cultivated rice has the AA genome (diploid, 2n = 24) and small genome size of only 430 megabase (haploid genome). As the sequencing of rice genome was completed by the International Rice Genome Sequencing Project (IRGSP), many researchers in the world have been working to explore the gene function on rice genome. Insertional mutagenesis has been a powerful strategy for assessing gene function. In maize, well characterized transposable elements have traditionally been used to clone genes for which only phenotypic information is available. In rice endogenous mobile elements such as MITE and Tos have been used to generate gene-tagged populations. To date T-DNA and maize transposable element systems have been utilized as main insertional mutagens in rice. The Ac/Ds system offers the advantage of generating new mutants by secondary transposition from a single tagged gene. To enhance the efficiency of gene detection, advanced gene-tagging systems (i.e. activation, gene or enhancer trap) have been employed for functional genomic studies in rice. Internationally, there have been many projects to develop large scales of insertional mutagenized populations and databases of insertion sites has been established. Ultimate goals of these projects are to supply genetic materials and informations essential for functional analysis of rice genes and for breeding using agronomically important genes. In this report, we summarize the current status of Ac/Ds-mediated gene tagging systems that has been conducted by collaborative works in Korea.

• BMB Reports
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• v.28 no.5
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• pp.458-463
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• 1995

Two apparent rho-independent transcription terminator structures within the coding sequence of aceK have been destroyed to access their roles in the differential expression between aceA and aceK in the glyoxylate bypass operon of E. coli. The effect of mutations on the expression of aceK was evaluated in two different ways: one by maxicell labeling and the other by lacZ fusion gene construction. The maxicell labeling experiment with the mutant operon clones has failed, like that of the wild type operon clone, to visibly show isocitrate dehrogenase (IDH) kinase/phosphatase, the product of aceK, on the autoradiogram of a protein gel. When the same mutations were introduced into an aceK::lacZ fusion gene to quantitatively evaluate the mutational effect, the activity of ${\beta}-galactosidase$ in neither of the mutant versions of the fusion gene was elevated significantly enough to explain the degree of polarity observed in this region. Thus, we conclude that neither of these intragenic, apparent rho-independent transcription terminator structures, which have long been suspected as a major determinant in the down regulation of aceK, really act as a premature transcriptional terminator.

• Journal of Microbiology
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• v.38 no.1
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• pp.18-23
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• 2000

Streptomyces coelicolor produces three kinds of catalases to cope with oxidative stress and to allow normal differentiation. Catalase A is the major vegetative catalase which functions in removing hydrogen peroxide generated during the process of aerobic metabolism. To understand the regulatory mechanism of response against oxidative stress, hydrogen peroxide-resistant mutant (HR4O) was isolated from S. coelicolor J1501 following UV mutagenesis. The mutant overproduced catalase A more than 50-fo1d compared with the wild type. The mutation locus catRI was mapped closed to the mthB2 locus by genetic crossings. An ordered cosmid library of S. coelicolor encompassing the mthB2 locus was used to isolate the regulator gene (catR) which represses catalase overproduction when introduced into HR4O. A candidate catR gene was found to encode a Fur-like protein of 138 amino acids (15319 Da).

• Journal of Microbiology
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• v.34 no.1
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• pp.15-22
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• 1996

The cloned X-bacterial gene (groEx) which is analogous to groE of E. Coli strongly expressed in E. coli when grown at the temperature 27.deg. C or higher without having to add any inducers. By S1-nuclease mapping, primer extension analysis and site directed mutagenesis, we found 4 promoters in the gene. Among them two promoters located at 5'-extended region of the gene are homologous to the promoters found in groE family of heat-shock genes ; they are , .sigma.$^{32}$ factor-dependent P1 promotor and .delta$^{70}$factor-dependet P2 promoter. The other two promoters found within the coding region of groESx were P3, 5'-TTGGCG-(18 bases)-AATACT-3' and P4, 5'-TTGGCA-(19 bases)-TAAGT which overlapped within 49 bases. These unique intragenic .delta.$^{70}$-dependent promoters are the first to be cloned and characterized in groE analogous heat-shock genes so far. These P3 and P4 promoters appeared to be responsible for the strong expression of GroElx in X-bacteria in vivo.

• Korean Journal of Microbiology
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• v.30 no.5
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• pp.347-354
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• 1992

New regulatory, loci which participate in the regulation of anaerobic inducible gene expression in Salmonella typhimurium were identified. We observed the regulatory network of new regulator mutations to various anaerobic inducible gene (1). Some anaerobic inducible lac fusions were also induced at low pH condition which was severe environment to withstand for its virulence at the place like phagolysosome. Sic oxygen-regulated regulatory mutants (oxr) isolated by Tn10 mutagenesis were divided into two groups. Five of them were found to show negative effect on the regulation of anaerobic gene expression, while on e showed positive effect on the regulation. Genetic loci of four oxr were identified with 54 Mud-P22 lysogens covering the whole chromosome of S. typhimurium, in the nearby region of map unit 87 min (oxr101), 63 min (oxr104), 97 min (oxr 105), and 57 min (oxr 106), respectively. Two oxr mutants were subjected to two-dimensional polyacrylamide electrophoretic analysis of anaerobic inducible proteins for searching the control circuitry of our oxr mutants.

• IMMUNE NETWORK
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• v.16 no.3
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• pp.176-182
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• 2016

CCR3 is a chemokine receptor that mediates the accumulation of allergic inflammatory cells, including eosinophils and Th2 cells, at inflamed sites. The regulatory sequence of the CCR3 gene, contains two Runt-related transcription factor (RUNX) 1 sites and two PU.1 sites, in addition to a functional GATA site for transactivation of the CCR3 gene. In the present study, we examined the effects of the cis-acting elements of RUNX1 and PU.1 on transcription of the gene in EoL-1 eosinophilic cells and Jurkat T cells, both of which expressed functional surface CCR3 and these two transcription factors. Introduction of RUNX1 siRNA or PU.1 siRNA resulted in a modest decrease in CCR3 reporter activity in both cell types, compared with transfection of GATA-1 siRNA. Cotransfection of the two siRNAs led to inhibition in an additive manner. EMSA analysis showed that RUNX1, in particular, bound to its binding motifs. Mutagenesis analysis revealed that all point mutants lacking RUNX1- and PU.1-binding sites exhibited reduced reporter activities. These results suggest that RUNX1 and PU.1 participate in transcriptional regulation of the CCR3 gene.

• KSBB Journal
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• v.11 no.4
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• pp.431-437
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• 1996

The nar promoter of Escherichta coli, which is maximally induced under anaerobic conditions in the presence of nitrate, was characterized to see whether the nar promoter cloned onto pBR322 can be used as an expression promoter. The modified nar promoter, in which several bases in the -10 region was mutated to the consensus sequence by site-directed mutagenesis, was characterized in E. coli, on which chromosome the fnr gene affecting expression of the nar promoter according to dissolved oxygen level was mutated. The E. coli lacZ gene was used as a reporter gene. The following effects were investigated to find optimal conditions to induce the modified nar promoter: induction methods, optimal nitrate concentrations, the amount of ${\beta}$-galactosidase expressed at the different growth conditions, and induction characteristics. The following results were obtained from the experiments : expression of ${\beta}$-galactosidase from the modified nar promoter was not affected much by nitrate concentrations. The maximal specific ${\beta}$-galactosidase activity was obtained when E. coli was grown under aerobic conditions, and then the modified nar promoter was induced at OD600=2.2 under microaerobic conditions (DO=1∼2%), under which conditions the maximal specific ${\beta}$-galactosidase activity was 13,000 Miller units. However, the specific ${\beta}$-galactosidase activity was approximately 6,000 Miller units even before the modified nar promoter was induced. Therefore, the modified nar promoter seemed to be useful when the cloned gene wants to be expressed in E. coli constitutively.

• Journal of Plant Biotechnology
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• v.45 no.3
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• pp.155-170
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• 2018

In this paper, mechanisms of gene editing technologies including ZFN, TALENS and CRISPR were briefly discussed with mutual advantages and disadvantages. Classification criteria of gene edited, site-directed mutagenesis (SDN) crops for regulatory purpose were also discussed. The number of studies using CRISPR technology was high and studies conducted on Arabidopsis thaliana and rice were highest, followed by tobacco, tomato, wheat, and corn. It has been applied to a variety of plants such as other grain crops, flower crops, vegetable crops, and fruit trees. The number of studies focused on practical application or commercialization in the future were also increasing yearly, and the scope of studies also expanded to include research on metabolic engineering for mass production of useful proteins or substances, development of disease resistant crops against viruses, bacteria, and fungi, abiotic environmental stressresistant crops, and increased yields. In addition to this, it was revealed that application range is becoming more diversified, including the development of parthenocarpic tomatoes, hybrid rice lines using male sterility and increased shattering resistance Brassica napus. It was also revealed that the number of CRISPR gene edited crops permitted by the USDA(APHIS) increases yearly, to be released in the international seed market soon.