• Microbiology and Biotechnology Letters
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• v.29 no.2
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• pp.72-77
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• 2001

Expression of f3 ~agarase Gene and Catabolite Repression in Escherichia coli by the Promoter of Alginate Lyase Gene Isolated from Marine Pseudomonas sp. Jin, Cheal~Ho, J~Hyeon Park, Jeong-Hyun Han, YoonM Hyeok Chae, Jong~Hee Lee, Jung-Kee Lee!, and In-800 Kong*. Faculty of Food Science and Biotechnology, Pukyong National UniversitYt Pusan 608-737, Korea, llnBioNet Co. 1690-3 Taejon 306-230, Korea - Promoter is a key factor for expression of the recombinant protein. There are many promoters for overexpression of protein in various organisms. The aly promoter of Pseudomonas sp. W7 isolated from marine environment was known to be a constitutive expression promoter of the alginate lyase gene, and it's promoter activity is repressed by glucose in Escherichia coli. To investigate the catabolite repression of the aly promoter ~md association between the promoter mutants, f3 agarase gene, which was also cloned from Pseudomonas sp. W7 was connected to the aly promoter with the sequence the coding 46 N-terminal amino acids ofthe alginate lyase gene. The constructed plasmid was introduced into E. coli and the agarase activity was measured. Fourty six amino acids of the alginate lyase gene was serially deleted using peR to the direction of 5' upstream region and subcloned. The agarase was overexpressed by the aly promoter and the production of agarase was repressed by the addition of glucose into culture media. Fourty six amino acids of alginate lyase did not affect the production of agarase at all. The deletion of a putative stem-loop structure in the aly promoter induced the decrease of f3 -agarase productivity.

• Journal of Life Science
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• v.20 no.10
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• pp.1451-1457
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• 2010

Serotonin (5-hydroxytryptamine, 5-HT) is an important mediator of cell-cell signaling in neuronal systems. The serotonin transporter (SERT) on the plasma membrane controls the extracellular 5-HT level by reuptake of released 5-HT from the synaptic cleft, but the underlying regulation mechanism is unclear. Here, we used the yeast two-hybrid system to identify the specific binding protein(s) that interacts with the carboxyl (C)-terminal region of SERT and found a specific interaction with protein kinase C-$\varepsilon$ (PKC-$\varepsilon$), a PKC isotype that is characterized as a calcium-independent and phorbol ester/diacylglycerol-sensitive serine/threonine kinase. PKC-$\varepsilon$ bound to the tail region of SERT but not to other members of the $Na^+/Cl^-$ dependent SLC6 gene family in the yeast two-hybrid assay. The C-terminal region of PKC-$\varepsilon$ is essential for interaction with SERT. In addition, these proteins showed specific interactions in the glutathione S-transferase (GST) pull-down assay. PKC-$\varepsilon$ phosphorylated the peptide of the SERT amino (N)-terminus in vitro. These results suggest that the phosphorylation of SERT by PKC-$\varepsilon$ may regulate SERT activity in plasma membrane.

• Korean Journal of Animal Reproduction
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• v.20 no.4
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• pp.453-458
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• 1997

It is known that the incorporation of genes into transgenic mice is generally stable and is p passed on to succeeding generations in a Mendelian fashion. In this report, transgenic mice were set as a model to evaluate whether the transgenes are transmitted in a Mendelian principle in a successive generations and how they are tran s smitted into their offspring. A 3.0 kb linear DNA fragment, containing the MMTV LTR, bovine aSI casein cDNA and SV 40 splicing and polyadenylation site; was microinjected into fertilized mouse embryos. The tail DNAs of the resulting pups were subjected to dot and Southern hybridizations to screen transgenic founders. The DNAs of their offspring were anlyzed by PCR to confirm the transmission of the transgene from F0. Out of 72 live pups four pups (5.6%), 3 males and 1 female, were positive for the transgene. The rates of transmission from F0 into F1 were 33.3, 7.7, 0, and 62.5%. Those from F1 into F2 were 63.6, 5.9, and 68.8% and those from F2 into F3 were 85.7, and 88.2%. In this report, the transmission pattern of transgenes in transgenic mice into their offspring was demonstrated. It either follows or does not follow in a Mendelian fashion. Deletion or loss of the transgenes from F0 in some lines became apparant to the succeeding generations.

• Clinical and Experimental Pediatrics
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• v.59 no.sup1
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• pp.25-28
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• 2016

Phelan-McDermid syndrome is a rare genetic disorder caused by the terminal or interstitial deletion of the chromosome 22q13.3. Patients with this syndrome usually have global developmental delay, hypotonia, and speech delays. Several putative genes such as the SHANK3, RAB, RABL2B, and IB2 are responsible for the neurological features. This study describes the clinical features and outcomes of Korean patients with Phelan-McDermid syndrome. Two patients showing global developmental delay, hypotonia, and speech delay were diagnosed with Phelan-McDermid syndrome via chromosome analysis, fluorescent in situ hybridization, and multiplex ligation-dependent probe amplification analysis. Brain magnetic resonance imaging of Patients 1 and 2 showed delayed myelination and severe communicating hydrocephalus, respectively. Electroencephalography in patient 2 showed high amplitude spike discharges from the left frontotemporoparietal area, but neither patient developed seizures. Kidney ultrasonography of both the patients revealed multicystic kidney disease and pelviectasis, respectively. Patient 2 experienced recurrent respiratory infections, and chest computed tomography findings demonstrated laryngotracheomalacia and bronchial narrowing. He subsequently died because of heart failure after a ventriculoperitoneal shunt operation at 5 months of age. Patient 1, who is currently 20 months old, has been undergoing rehabilitation therapy. However, global developmental delay was noted, as determines using the Korean Infant and Child Development test, the Denver developmental test, and the Bayley developmental test. This report describes the clinical features, outcomes, and molecular genetic characteristics of two Korean patients with Phelan-McDermid syndrome.

• Journal of Life Science
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• v.21 no.12
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• pp.1778-1783
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• 2011

Hematopoietic cytokines regulate production of blood cells by stimulating proliferation and differentiation of bone marrow cells. Among these hematopoietic cytokines, called hematopoitic growth factors, glranulocyte-colony stimulating Factor (G-CSF), which regulates growth of neutrophils, is one of important therapeutic factors because cancer patients suffer with neutropenia which is severe reduction of neutrophils after chemotherapy. Two groups of recombinant G-CSF have approved and used for therapeutic purposes and many researches are still on-going to produce recombinant G-CSF by different techniques. We engineered human G-CSF with Bombyx specific endoplasmic reticulum (ER) signal sequence, therefore, secretion of human G-CSF protein was improved in Bombyx mori-origined cell line, Bm5. The Bombyx ER signal sequence and human G-CSF matured protein region chimera was further remodeled at the N-terminus of matured G-CSF protein to understand roles of N-terminus on outer cellular secretion and/or production. Three different mutants were generated deleting three amino acids in non alpha-helical region in N-terminus in order to scan important amino acids for G-CSF secretion. One of 3 different N-terminal deletion mutants showed dramatically reduction of secreted amount of G-CSF indicating its important role on secretion. The data suggest that remodeling in non alpha-helical region of N-terminus is also important for recombinant G-CSF production.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.54-54
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• 2001

Human chorionic gonadotropin (hCG) is a member of the glycoprotein hormone family which includes FSH, hCG, TSH. These hormone family is characterized by a heterodimeric structure composed a common $\alpha$-subunit noncovalently linked to a hormone specific $\beta$-subunit. The correct conformation of the heterodimer is also important for efficient secretion, hormone-specific post-translational modifications, receptor binding and signal transduciton. To determine $\alpha$ and $\beta$-subunits can be synthesized as a single polypeptide chain (tethered-hCG) and also display biological activity, the tethered-hCG molecule by fusing the carboxyl terminus of the hCG $\beta$-subunit to the amino terminus of the $\alpha$-subunit was constructed and transfected into chinese hamster ovary (CHO-K1) cells. We also constructed C-terminal deletion mutants (D9l, D89, D88, D87, D86, D84, D83) of single chain hCG to determine the biological function (secretion, LH-activity, receptor binding, cAMP production) of these mutants. Between six and eight stably transfected pools of cells expressing wild type and mutant hCGs were selected for neomycin resistant. The hCGs secreted by the stably transfected cells into serum-free media were collected and quantified by radioimmunoassay, as described in protocol (DPC(hCG IRMA). LH activity was in terms of testosterone production and aromatase activity in primary cultured rat Leydig cells. The tethered-wthCG was efficiently secreted and showed similar LH-like activity to the dimeric hCG. The D83hCG mutant was not detected in this assay. It is suggest that hCG C-terminal part is very important for hCG secretion. Now, we checking the LH-like activity of these mutant hCGs. These data indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion.

• BMB Reports
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• v.43 no.3
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• pp.193-198
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• 2010

In this study, we report the identification and characterization of a novel C2H2 zinc finger protein, ZNF552, from a human embryonic heart cDNA library. ZNF552 is composed of three exons and two introns and maps to chromosome 19q13.43. The cDNA of ZNF552 is 2.3 kb, encoding 407 amino acids with an amino-terminal KRAB domain and seven carboxyl-terminal C2H2 zinc finger motifs in the nucleus and cytoplasm. Northern blotting analysis indicated that a 2.3 kb transcript specific for ZNF552 was expressed in liver, lung, spleen, testis and kidney, especially with a higher level in the lung and testis in human adult tissues. Reporter gene assays showed that ZNF552 was a transcriptional repressor, and overexpression of ZNF552 in the COS-7 cells inhibited the transcriptional activities of AP-1 and SRE, which could be relieved through RNAi analysis. Deletion studies showed that the KRAB domain of ZNF552 may be involved in this inhibition.

• Journal of Physiology & Pathology in Korean Medicine
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• v.20 no.3
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• pp.651-656
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• 2006

Activation of $NF-{\kappa}B$ is known to be a trigger of various cellular disorders including inflammatory and autoimmune diseases such as rheumatoid arthritis. Numerous approaches are ongoing within laboratories to identify potential therapeutic agents which inhibit the $NF-{\kappa}B$ activation. In this study, we have tested the inhibitory effects of five traditional medicines on the activation of $NF-{\kappa}B$ by NIK. Among three medicines which exhibited inhibitory effect on the expression of $NF-{\kappa}B$ repoter plasmid, we investigated further the inhibitory mechanism of Dichroa febrifuga in connection with IKKY activity. Wild type $IKK{\gamma}$ inhibited the $NF-{\kappa}B$ activation by NIK but the C-terminal deletion mutant of IKKY did not show the inhibitory effect, indicating that the C-terminal leucine zipper domain of $NF-{\kappa}B$ is important for the inhibition of $NF-{\kappa}B$ activation. The water extract of Dichroa febrifuga(DFE) also strongly inhibited the $NF-{\kappa}B$ activation by NIK. The inhibitory activity of DFE appeared to be independent of the expression of $IKK{\gamma}$, suggesting that the pathways of inhibition by Dichroa febrifuga and $IKK{\gamma}$ are different. Our results suggest that Dichroa febrifuga can be used as a medicine for inhibition of the $NF-{\kappa}B$ activation in a wide range of cells without relation to the expression of $IKK{\gamma}$.

• Proceedings of the Botanical Society of Korea Conference
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• 2002.04a
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• pp.62-72
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• 2002

This study has investigated the biosynthesis and function of the heavy metal binding peptides, the phytochelatins, in plants. PCs are synthesised enzymatically from glutathione by the enzyme PC synthase in the presence of heavy metal ions. Using Arabidopsis thaliana as a model organism cadmium-sensitive, phytochelatin-deficient mutants have been isolated and characterised in previous studies. The cadl mutants have wildtype levels of glutathione, are PC deficient and lack PC synthase activity. Thus, the CADl gene has been proposed to encode PC synthase. The CADl gene was isolated by a positional cloning strategy The gene was mapped and a candidate identified. Each of four cadl mutants had a single base pair change in the candidate gene and the cadmium-sensitive, cadl phenotype was complemented by the candidate gene. This demonstrated the CADl gene had been cloned. A homologous gene in the fission yeast, Schizosaccharomyces pombe was identified through database searches. A targeted-deletion mutation of this gene was constructed and the mutant, like cadl mutants of Arabidopsis, was cadmium-sensitive and PC-deficient. A comparison of the redicted amino acid sequences reveals a highly conserved N-terminal region Presumed to be the catalytic domain and a variable C-terminal region containing multiple Cys residues proposed to be involved in activation of the enzyme by metal ions. Similar genes were also identified in animal species. The Arabidopsis CADl/AtPCSl and S. pombe SpbPCS genes were expressed in E. coli and were shown to be sufficient for glutathione-dependent, heavy metal activate PC synthesis in vitro, thus demonstrating these genes encode PC synthase enzymes. Using RT-PCR, AtPCSl expression appeared to be independent of Cd exposure. However, at higher levels of Cd exposure a AtPCSl-CUS reporter gene construct appeared to be more highly expressed. Using the reporter gene construct, AtPCSl was expressed most tissues. Expression appeared to be greater in younger tissues and same higher levels of expression was observed in some regions, including carpels and the base of siliques. AtPCS2 was a functional gene encoding an active PC synthase. However, its Pattern of expression and the phenotype of a mutant (or antisense line) have not been determined. Assuming the gene is functional then it has clearly been maintained through evolution and must provide some selective advantage. This implies that, at least in some cells or tissue, it is likely to be the dominant PC synthase expressed. This remains to be determined

• Korean Journal of Microbiology
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• v.28 no.1
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• pp.13-18
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• 1990

A DNA sequence encoding the adr subtype preS2 region of hepatitis B virus envelope protein was fused to 5' end of lacZ gene yielding a plasmid pTSZ, in order to produce a preS2-$\beta$-galactosidase fusion protein. Serial deletions from 3' and 5' end of preS2 were constructed in plasmids, which were expressed and their antigenicities were examined with the monoclonal antibody H8. Deletions from amino and carboxy terminal to certain points did not affect the antigenicity, but the longer deletions destroyed the antigenicity. End points of deleted preS2 sequence were determined by DNA sequencing. As a result, each end of preS2 epitope was located in the region of amino acid residue 130-132 and 140-142, respectively. Residue 143 may be supplementary for antigenic epitope since the deletion from carboxy terminal to residue 143 revealed partial defect of antigenicity. In the interval of antigenic epitope the amino acid differences between adr and adw2 subtype occurred ar residue 130, 132, and 141. This result indicated that one or more of the three residues are responsible for the binding specificity of monoclonal antibody H8 to adr subtype preS2 fusion protein.