• Molecules and Cells
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• v.27 no.5
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• pp.497-502
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• 2009

In mouse embryos, somite formation occurs every two hours, and this periodic event is regulated by a biological clock called the segmentation clock, which involves cyclic expression of the basic helix-loop-helix gene Hes7. Hes7 expression oscillates by negative feedback and is cooperatively regulated by Fgf and Notch signaling. Both loss of expression and sustained expression of Hes7 result in severe somite fusion, suggesting that Hes7 oscillation is required for proper somite segmentation. Expression of a related gene, Hes1, also oscillates by negative feedback with a period of about two hours in many cell types such as neural progenitor cells. Hes1 is required for maintenance of neural progenitor cells, but persistent Hes1 expression inhibits proliferation and differentiation of these cells, suggesting that Hes1 oscillation is required for their proper activities. Hes1 oscillation regulates cyclic expression of the proneural gene Neurogenin2 (Ngn2) and the Notch ligand Delta1, which in turn lead to maintenance of neural progenitor cells by mutual activation of Notch signaling. Taken together, these results suggest that oscillatory expression with short periods (ultradian oscillation) plays an important role in many biological events.

• The Plant Pathology Journal
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• v.25 no.1
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• pp.47-53
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• 2009

Sequence analysis of the Chlorella virus SS-2 revealed one putative nuclease gene that is 807 bp long and encodes a 31kDa protein. Multiple sequence alignment analysis reveals the presence of highly conserved PD-(D/E)XK residues in the encoded protein. The gene cloned into an expression vector was expressed as a His-tagged fusion protein in chaperone containing pKJE7 cells. The recombinant protein was purified using a His-Trap chelating HP column and used for functional analysis. Exonuclease activity of the SS-2 nuclease was detected when the DNA substrates, such as linear ssDNA, PCR amplicon, linear dsDNA with 5'-overhang ends, 3'-overhang ends, or blunt ends were used. Covalently closed circular DNA was also degraded by the SS-2 recombinant protein, suggesting that the SS-2 nuclease has an endonuclease activity. Stable activity of SS-2 nuclease was observed between $10^{\circ}C$ and $50^{\circ}C$. The optimum pH concentrations for the SS-2 nuclease were pH 6.0-8.5. Divalent ions inhibited the SS-2 nuclease activity.

• Proceedings of the Korean Society of Crop Science Conference
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• 2002.05a
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• pp.48-50
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• 2002

A strategy for development of a functional rice in proved with human lactoferrin and enhancement of nutrient compounds was planned. For the purposes we have cloned and characterized a human lactoferrin cDNA from human mammary gland cDNA library A endosperm storage vacuole targeting sequence and the cDNA fragment was linked to endosperm specific glutelin promoter. The fusion gene fragment was inserted into a binary vector containing MAR gene. In addition a new ${\beta}$-galactosidase gene from Bifidobacterium of human was used as a reporter gene in the vector system, Rice plants showing a high concentration of amino acids in the endosperm cells were developed by using a biochemical mutation and bred for the transformation with the binary vector system Finally we have established a transformation method for the rice endosperm cells.

• International Journal of Industrial Entomology and Biomaterials
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• v.17 no.2
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• pp.223-228
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• 2008

A novel beetle antimicrobial protein from stimulated Copris tripartitus and the corresponding gene were isolated in parallel through differential display-PCR and expression in Escherichia coli. To find cDNA clones responsible for bacteria resistance, the suppression subtractive hybridization and GeneFishing differentially expressed genes system were employed in the dung beetle, Copris tripartitus immunized with lipopolysaccaride. One cDNA clone from eight subtracted clones was selected through dot blot analysis and confirmed by northern blot analysis. The 516-bp, selected cDNA clone was determined by 5' and 3' rapid amplication of cDNA ends and cloned into the GST fusion expression vector pGEX-4T-1 for expression of the protein. The expressed protein was predicted 14.7 kDa and inhibited the growth of gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa. These results implied that the expressed protein is related to immune defense mechanism against microorganism.

• BMB Reports
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• v.29 no.1
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• pp.92-97
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• 1996

The age-46.5 gene of Escherichia coli is induced by nitrate ion and regulated by Fnr, NarL, and NarP during anaerobic growth. aeg-46.5::lacZ fusion gene shows its maximum expression in narL host after two hours of aerobic to anaerobic switch in M9-Glc-nitrate medium. Fnr and NarP act as positive regulators, and NarL acts as a negative regulator. The control region of the aeg-46.5 was identified and the binding sites of regulator proteins have been predicted (Reznikoff and Choe (1993)). It has two symmetry regions. One is located at -52~-37 bp from the anaerobic mRNA 5'-end, which is the binding site of NarL and NarP. The other is located at +37~+56 bp from the 5'-end of mRNA. In this study, the downstream symmetry region from the mRNA 5'-end was investigated by site-directed mutagenesis. The destruction of the symmetry region increases the expression level of aeg-46.5. We propose that the symmetry region interferes with the expression of aeg-46.5 possibly by forming a stem-and-loop structure.

• BMB Reports
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• v.35 no.6
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• pp.562-567
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• 2002

In order to find the cellular interaction factors of the Heliothis armigera nuclear polyhedrosis virus capsid protein VP39, a Heliothis armigera cell cDNA library was constructed. Then VP39 was used as bait. The host actin gene was isolated from the cDNA library with the yeast two-hybrid system. This demonstrated that VP39 could interact with its host actin in yeast. In order to corroborate this interaction in vivo, the vp39 gene was fused with the green fluorescent protein gene in plasmid pEGFP39. The fusion protein was expressed in the Hz-AM1 cells under the control of the Autographa californica multiple nucleopolyhedrovirus immediate early gene promoter. The host actin was labeled specifically by the red fluorescence substance, tetramethy rhodamine isothicyanete-phalloidin. Observation under a fluorescence microscopy showed that VP39, which was indicated by green fluorescence, began to appear in the cells 6 h after being transfected with pEGFP39. Red actin cables were also formed in the cytoplasm at the same time. Actin was aggregated in the nucleus 9 h after the transfection. The green and red fluorescence always appeared in the same location of the cells, which demonstrated that VP39 could combine with the host actin. Such a combination would result in the actin skeleton rearrangement.

• Molecules and Cells
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• v.26 no.6
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• pp.554-557
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• 2008

Double-stranded RNA (dsRNA) induces gene silencing in a sequence-specific manner by a process known as RNA interference (RNAi). The RNA-induced silencing complex (RISC) is a multi-subunit ribonucleoprotein complex that plays a key role in RNAi. VIG (Vasa intronic gene) has been identified as a component of Drosophila RISC; however, the role VIG plays in regulating RNAi is poorly understood. Here, we examined the spatial and temporal expression patterns of VIG-1, the C. elegans ortholog of Drosophila VIG, using a vig-1::gfp fusion construct. This construct contains the 908-bp region immediately upstream of vig-1 gene translation initiation site. Analysis by confocal microscopy demonstrated GFP-VIG-1 expression in a number of tissues including the pharynx, body wall muscle, hypodermis, intestine, reproductive system, and nervous system at the larval and adult stages. Furthermore, western blot analysis showed that VIG-1 is present in each developmental stage examined. To investigate regulatory sequences for vig-1 gene expression, we generated constructs containing deletions in the upstream region. It was determined that the GFP expression pattern of a deletion construct (${\Delta}-908$ to -597) was generally similar to that of the non-deletion construct. In contrast, removal of a larger segment (${\Delta}-908$ to -191) resulted in the loss of GFP expression in most cell types. Collectively, these results indicate that the 406-bp upstream region (-596 to -191) contains essential regulatory sequences required for VIG-1 expression.

• BMB Reports
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• v.31 no.1
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• pp.77-82
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• 1998

To purify the geranylgeranyl protein transferase type I (GGPT-I) efficiently, a gene expression system using the pGEX-4T-1 vector was constructed. The cal1 gene, encoding the ${\beta}$ subunit of GGPT-I, was subcloned into the pGEX-4T-1 vector and co-transformed into E. coli cells harboring the ram2 gene, the ${\alpha}$ subunit gene of GGPT-I. GGPT-I was highly expressed as a fusion protein with glutathione S-transferase (GST) in E. coli, purified to homogeneity by glutathione-agarose affinity chromatography, and the GST moiety was excised by thrombin treatment. The purified yeast GGPT-I showed a dose-dependent increase in the transferase activity, and its apparent $K_m$ value for an undecapeptide fused with GST (GST-PEP) was $0.66\;{\mu}M$ and the apparent value for geranylgeranyl pyrophosphate (GGPP) was $0.071\;{\mu}M$.

• The Journal of Korean Society of Virology
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• v.27 no.1
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• pp.9-17
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• 1997

Hepatitis C virus (HCV) E2 protein is known to be one of putative envelope proteins. To develop a sensitive detection method for HCV infected tissues and cells, monoclonal antibodys (MAbs) to the E2 protein of HCV were prepared from mice immunized with recombinant baculovirus-expressing E2 protein (Bac-E2). Several hybridoma clones secreting various levels of MAb were isolated and isotypes of these MAb were determined. One clone (L.2.3.3) was used for ascites production and the E2-MAb was purified and characterized. The L.2.3.3 reacted well with both Bac-E2 and E. coli expressed glutathione-S-transferase-E2 (GST-E2) fusion proteins. Using HCV patient sera, E2 envelope protein was found to be localized in the cell membrane boundary both in CHO cells and insect cells which express HCV E2 protein. Similar result was obtained when same cells were treated with the MAb L.2.3.3. These results demonstrated that Bac-E2 protein is capable of eliciting high titer antibody production in mice.

• 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.