• Korean Journal of Microbiology
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• v.47 no.1
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• pp.1-6
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• 2011

Inefficiency of in vivo gene transfer using currently available vectors reflects a major hurdle in cancer gene therapy. Both viral and non-viral approaches have been described to improve gene transfer efficiency but suffer from a number of limitations. Here we tested an adenovirus carrying the small peptide ligand derived from heregulin${\beta}$ EGF-like domain onto fiber, the adenoviral capsid protein, to deliver transgene to ovarian cancer cells which overexpress ErbB, the cognate receptors for heregulin. The attachement of 53 amino acids to fiber didn't affect on the fiber's trimer structure that is critical for the viral entry to cells. The fiber-modified adenovirus can mediate entry and expression of a ${\beta}$-galactosidase into cancer cells in an increased efficiency compared the unmodified adenovirus. Particularly, the gene transfer efficiency was improved up to 5 times in OVCAR3 cells, an ovarian cancer cell line. Such transduction systems hold promise for delivering genes to ErbB receptor overexpressing cancer cells, and could be used for future cancer gene therapy.

• BMB Reports
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• v.37 no.1
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• pp.107-113
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• 2004

Since the completion of the genome project of the nematode C. elegans in 1998, functional genomic approaches have been applied to elucidate the gene and protein networks in this model organism. The recent completion of the whole genome of C. briggsae, a close sister species of C. elegans, now makes it possible to employ the comparative genomic approaches for identifying regulatory mechanisms that are conserved in these species and to make more precise annotation of the predicted genes. RNA interference (RNAi) screenings in C. elegans have been performed to screen the whole genome for the genes whose mutations give rise to specific phenotypes of interest. RNAi screens can also be used to identify genes that act genetically together with a gene of interest. Microarray experiments have been very useful in identifying genes that exhibit co-regulated expression profiles in given genetic or environmental conditions. Proteomic approaches also can be applied to the nematode, just as in other species whose genomes are known. With all these functional genomic tools, genetics will still remain an important tool for gene function studies in the post genome era. New breakthroughs in C. elegans biology, such as establishing a feasible gene knockout method, immortalized cell lines, or identifying viruses that can be used as vectors for introducing exogenous gene constructs into the worms, will augment the usage of this small organism for genome-wide biology.

• BMB Reports
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• v.34 no.1
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• pp.51-58
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• 2001

Attempts using in vitro and in vivo selection procedures have been made to search for hammerhead ribozymes that have higher activities than the wild-type ribozyme and also to determine whether other sequences might be possible in the catalytic core of the hammerhead ribozyme. Active sequences selected in the past conformed broadly to the consensus core sequence except at A9, and no sequences were associated with higher activity than that of the hammerhead with the consensus core, an indication that the consensus sequence derived from viruses and virusoids is probably the optimal sequence [Vaish et al. (1997) Biochemistry 36, 6495-6501]. Recently, during construction of ribozyme expression vectors, we isolated a mutant hammerhead ribozyme, with an insertion of G between A9 and G10.1, that appeared to show significant activity [Kawasaki et al. (1996) Nucleic Acids Res. 24, 3010-3016; Kawasaki et al. (1998) Nature 393, 284-289]. We, therefore, characterized kinetic properties of the G-inserted mutant ribozymes in terms of the NUX rule. We demonstrate that the NUX rule is basically applicable to the G-inserted ribozymes and, more importantly, one type of G-inserted ribozyme was very active with $k_{cat}$, value of $6.4\;min^{-1}$ in 50 mM Tris-HCl (pH 8.0) and 10 mM $MgCl_2$ at $37^{\circ}C$.

• BMB Reports
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• v.51 no.7
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• pp.338-343
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• 2018

Transcription termination factor-1 (TTF-I) is an RNA polymerase 1-mediated transcription terminator and consisting of a C-terminal DNA-binding domain, central domain, and N-terminal regulatory domain. This protein binds to a so-called 'Sal box' composed of an 11-base pair motif. The interaction of TTF-I with the 'Sal box' is important for many cellular events, including efficient termination of RNA polymerase-1 activity involved in pre-rRNA synthesis and formation of a chromatin loop. To further understand the role of TTF-I in human immunodeficiency virus (HIV)-I virus production, we generated various TTF-I mutant forms. Through a series of studies of the over-expression of TTF-I and its derivatives along with co-transfection with either proviral DNA or HIV-I long terminal repeat (LTR)-driven reporter vectors, we determined that wild-type TTF-I downregulates HIV-I LTR activity and virus production, while the TTF-I Myb-like domain alone upregulated virus production, suggesting that wild-type TTF-I inhibits virus production and trans-activation of the LTR sequence; the Myb-like domain of TTF-I increased virus production and trans-activated LTR activity.

• Development and Reproduction
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• v.14 no.2
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• pp.43-50
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• 2010

Recently induced pluripotent stem (iPS) cells are derived from somatic cells by ectopic expression of several transcription factors (reprogramming factors) using technology of somatic cell reprogramming. iPS cells are able to selfrenew and differentiate into all type of cells in the body similarly to embryonic stem cells. Because iPS cells have advantages that can avoid immune rejection after transplantation and ethical issues unlike embryonic stem cells, research on iPS has made significant progress since the first report by Yamanaka in 2006. Nevertheless of many advantages of iPS, safer methods to introduce reprogramming factors into somatic cells must be developed due to safety concerns regarding viral vectors, and safer reprogramming factors to substitute the oncogenes should be evaluated for clinical application of iPS. Here we discuss the recent progress in reprogramming factors in embryonic stem cells, oocytes, and embryos, and discuss further research for finding new, more reliable and safer reprogramming factors.

• Journal of Microbiology and Biotechnology
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• v.14 no.5
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• pp.1052-1056
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• 2004

A secretion signal sequence-selection vector (pGS40) was constructed based on an $\alpha$-amylase gene lacking a secretion signal and employed for selecting secretion signals from Lactococcus lactis ssp. cremoris LM0230 chromosomal DNA. Six fragments were identified based on their ability to restore $\alpha$-amylase secretion in E. coli, and among these, a fragment, S405, conferred the highest secretion activity (84%) in E. coli. Meanwhile, S407, which conferred poor secretion activity in E. coli, was quite active in L. lactis. The results suggested that the efficiency of a secretion signal depended on the host. All six fragments had an open reading frame (ORF) fused to the reporter gene, and the potential Shine-Dalgamo (SD) sequence and putative promoter sequences were located upstream of the ORF. Deduced amino acid sequences from the six fragments did not show any homology with known secretion signals. However, they contained three distinguished structural features and cleavage sites, commonly found among typical secretion signals. The characterized secretion signals could be useful for the construction of food-grade secretion vectors and gene expression in LAB.

• Journal of Applied Biological Chemistry
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• v.53 no.4
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• pp.189-194
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• 2010

We confirmed the hypo-osmotic shock strengths and duration, different type of vectors, and subcelluar localization to identify the optimum analysis condition of plant aquaporin activity in Xenopus ooctye using Arabidopsis thaliana AtPIP2-1 gene. Six minutes and 1/5ND buffer hypoosmotic shock treatment was the best condition to show the maximum swelling of Xenopus oocytes where AtPIP2-1 was expressed using pcDNA3.1 vector. AtPIP2-1 protein was expressed more efficiently in pGEMHE vector which has 5' and 3' UTR (untranslation region) of Xenopus ${\beta}$-GLOBIN gene in multiple cloning site than in pcDNA3.1 vector. Also green fluorescence of GFP fused to AtPIP2-1 was detected onto oocyte plasmamembrane where is the proper subcellular localization target of AtPIP2-1.

• Reproductive and Developmental Biology
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• v.36 no.3
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• pp.207-212
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• 2012

The shortage of human organs for transplantation has induced the research on the possibility of using animal as porcine. However, pig to human transplantation as known as xeno-transplantation has major problem as immunorejection. Recently, the solutions of pig to human xenotransplantation are commonly mentioned as having a genetically modification which include alpha 1, 3 galatosyl transferase knockout (GTKO) and immune-suppressing gene transgenic model. Unfortunately, the expression level of transgenic gene is very low activity. Therefore, development of gene overexpression system is the most urgent issue. Also, the tissue specific overexpression system is very important. Because most blood vessels are endothelial cells, establishment of the endothelial-specific promoter is attractive candidates for the introduction of suppressing immunorejection. In this study, we focus the ICAM2 promoter which has endothelial-specific regulatory region. To detect the regulatory region of ICAM2 promoter, we cloned 3.7 kb size mini-pig ICAM2 promoter. We conduct serial deletion of 5' flanking region of mini-pig ICAM2 promoter then selected promoter size as 1 kb, 1.5 kb, 2 kb, 2.5 kb, and 3 kb. To analyze promoter activity, luciferase assay system was conducted among these vectors and compare endothelial activity with epithelial cells. The reporter gene assay revealed that ICAM2 promoter has critical activity in endothelial cells (CPAE) and 1 kb size of ICAM2 promoter activity was significantly increased. Taken together, our studies suggest that mini-pig ICMA2 promoter is endothelial cell specific overexpression promoter and among above all size of promoters, 1 kb size promoter is optimal candidate to overcome the vascular immunorejection in pig to human xenotransplantation.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.563-569
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• 2004

The DNA sequence of the chitosanase gene (choK) from $\beta$-Proteobacterium KNU3 showed an 1,158-bp open reading frame that encodes a protein of 386 amino acids with a novel 74 signal peptide. The degenerated primers based on the partial deduced amino acid sequences from MALDI- TOF MS analyses yielded the 820 bp of the PCR product. Based on this information, double inverse PCR cloning experiments, which use the two specific sets of PCR primers rather than single set primers, identified the unknown 1.2 kb of the choK gene. Subsequently, a 1.8 kb of full choK gene was cloned from another PCR cloning experiment and it was then subcloned into pGEM T-easy and pUC18 vectors. The recombinant E. coli clone harboring recombinant pUC18 vector produced a clear halo around the colony in the glycol chitosan plates. The recombinant ChoK protein was secreted into medium in a mature form while the intracellular ChoK was produced without signal peptide cleavage. The activity staining of PAGE showed that the recombinant ChoK protein was identical to the chitosanase of wild-type. The comparison of deduced amino acid sequences of choK revealed that there is 92% identity with that of Sphingobacterium multivorum chitosanase. Judging from the conserved module in other bacterial chitosanases, chitosanase of KNU3 strain (ChoK) belongs to the family 80 of glycoside hydrolases.

• Journal of Microbiology and Biotechnology
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• v.2 no.4
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• pp.243-247
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• 1992

The thr operon of Escherichia coli TF427, an $\alpha$-amino-$\beta$-hydroxyvaleric acid (AHV)-resistant threonine overproducer, was cloned in a pBluescriptII $KS^＋$ plasmid by complementation of E. coli mutants. All clones contained a common 8.8 kb HindIII-generated DNA fragment and complemented the thrA, thrB, and thrC mutants by showing that these clones contained the whole thr operon. This thr operon was subcloned in the plasmid vectors pBR322, pUC18, and pECCG117, an E. coli/Corynebacterium glutamicum shuttle vector, to form recombinant plasmids pBTF11, pUTF25 and pGTF18, respectively. The subcloned thr operon was shown to be present in a 6.0 kb insert. A transformant of E. coli TF125 with pBTF11 showed an 8~11 fold higher aspartokinase I activity, and 15~20 fold higher L-threonine production than TF125, an AHV-sensitive methionine auxotroph. Also, it was found that the aspartokinase I activity of E. coli TF125 harboring pBTF11 was not inhibited by threonine and its synthesis was not repressed by threonine plus isoleucine.