• Journal of Microbiology and Biotechnology
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• v.7 no.4
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• pp.229-236
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• 1997

A gene, $phbC_{2.4.1}$ encoding poly-3-hydroxybutyric acid (PHB) synthase of Rhodobacter sphaeroides 2.4.1 was cloned by employing heterologous expression in Escherichia coli. R. sphaeroides chromosomal DNA partially digested with MboI was cloned in pUC19 followed by mobilization into E. coli harbouring $phbA,B_{AC}$ in pRK415, which code for ${\beta}$-ketothiolase and acetoacetyl CoA reductase of Alcaligenes eutrophus, respectively. Two E. coli clones carrying R. sphaeroides chromosomal fragment of $phbC_{2.4.1}$ in pUC19 were selected from ca. 10,000 colonies. The PHB-producing colonies had an opaque white appearance due to the intracellular accumulation of PHB. The structure of PHB produced by the recombinant E. coli as well as from R. sphaeroides 2.4.1 was confirmed by [$H^{+}$]-nuclear magnetic resonance (NMR) spectroscopy. Restriction analysis of the two pUC19 clones revealed that one insert DNA fragment is contained as a part of the other cloned fragment. An open reading frame of 601 amino acids of $phbC_{2.4.1}$ with approximate M.W. of 66 kDa was found from nucleotide sequence determination of the 2.8-kb SaiI-PstI restriction endonuclease fragment which had been narrowed down to support PHB synthesis through heterologous expression in the E. coli harbouring $phbA,B_{AC}$. The promoter (s) of the $phbC_{2.4.1}$ were localized within a 340-bp DNA region upstream of the $phbC_{2.4.1}$ start codon according to heterologous expression analysis.

• Journal of Life Science
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• v.30 no.3
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• pp.285-290
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• 2020

Foot-and-mouth disease virus (FMDV), a member of the genus Aphthovirus in the Picornaviridae family, affects wild and domesticated ruminants and pigs. FMDV causes various clinical symptoms, including severe inflammation in infected tissue. Genome RNA of FMDV shows a positive single-strand chain approximately 8.3 kb long and encodes a single long open reading frame (ORF). The ORF is translated into structural and non-structural proteins by viral proteases. The FMDV 2C protein is one of the non-structural proteins encoded by FMDV and plays a critical role in FMD pathogenesis, including inflammation, apoptosis, and viral replication. In this study, we examined whether FMDV 2C induces intracellular expression of pro-inflammatory cytokine tumor necrosis factor alpha (TNFα). FMDV 2C expression in pig IBRS-2 cells increased mRNA and protein expression of TNFα at the transcriptional level via activation of TNFα promoter. Treatment with 4-phenylbutyric acid, an endoplasmic reticulum (ER) stress reducer, decreased TNFα expression induced by FMDV 2C. Activating transcription factor 4 (ATF4), a transcription factor mediating ER stress response, induced transactivation of TNFα promoter and expression of mRNA and protein of TNFα. However, the dominant negative mutant of ATF4 did not induce FMDV 2C-mediated TNFα expression. The results indicate that FMDV 2C protein increases clinical inflammation via ATF4-mediated TNFα expression and is associated with ER stress induction.

• Journal of Microbiology and Biotechnology
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• v.6 no.2
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• pp.145-146
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• 1996

The 2.4-kb cryptic plasmid (pKH8) of multidrug-resistant Staphylococcus aureus SA2 was characterized by complete nucleotide sequencing and homology comparison. pKH8 was found to contain three open reading frames. Protein analysis of pKH8 showed that pKH8 was a multidrug resistance plasmid and mediated resistance to ethidium bromide and quaternary ammonium compounds.

• Proceedings of the Mineralogical Society of Korea Conference
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• 2002.10a
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• pp.119-136
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• 2002

Contrasts in the style of the gold-silver mineralization in geologic and tectonic settings in Korea, together with radiometric age data, reflect the genetically different nature of hydrothermal activities, coinciding with the emplacement age and depth of Mesozoic magmatic activities. It represents a clear distinction between the plutonic settings of the Jurassic Daebo orogeny and the subvolcanic environments of the Cretaceous Bulgugsa igneous activities. During the Daebo igneous activities (c.a. 200-150 (?) Ma) coincident with orogenic time, gold mineralization took place between c.a. 195 and 135 (127 ?) Ma. The Jurassic Au deposits commonly show several characteristics; prominent association with pegmatites, low Ag/hu ratios in the ore-concentrating parts, massive vein morphology and a distinctively simple mineralogy including Fe-rich sphalerite, galena, chalcopyrite, arsenopyrite, Au-rich electrum, pyrrhotite and/or pyrite. During the Bulgugsa igneous activities $(110\~50Ma)$, the precious-metal deposits are generally characterized by such features as complex vein morphology, medium to high AE/AU ratios in the ore concentrates, and diversity of ore minerals including base-metal sulfides, pyrite, arsenopyrite, Ag-rich electrum and native silver nth Ag sulfides, Ag-Sb-As sulfosalts and Ag tellurides. Vein morphology, mineralogical, fluid inclusion and stable isotope results indicate the diverse genetic natures of hydrothermal systems in Korea. The Jurassic Au-dominant deposits (orogenic type) were formed at the relatively high temperature $(about\;300^{\circ}\;to\;450^{\circ}C)$ and deep-crustal level $(4.0{\pm}1.5\;kb)$ from the hydrothermal fluids containing more amounts of magmatic waters $(\delta\;^{18}O_{H2O}\;5\~10\%_{\circ})$. It can. It can be explained by the dominant ore-depositing mechanisms as $CO_2$ boiling and sulfidation, suggestive of hypo- to mesothermal environments. In contrast, the Cretaceous Au-dominant $(l13\~68\;Ma),\;Au-Ag \;(108\~47\;Ma)$ and Ag-dominant $(103\~45\;Ma)$ deposits, which correspond to volcanic-plutonic-related type, occurred at relatively low temperature $(about\;200^{\circ}\;to\;350^{\circ}C)$ and shallow-crustal level $(1.0\{pm}0.5\;kb)$ from the ore-forming fluids containing more amounts of less-evolved meteoric waters$(\delta\;^{18}O_{H2O}\;-10\~5\%_{\circ})$. These characteristics of the Cretaceous precious-metal deposits can be attributed to the complexities in the ore-precipitating mechanisms (mixing, boiling, cooling), suggestive of epi- to mesothermal environments. Therefore, the differences of the emplacement depth between the Daebo and the Bulgugsa igneous activities directly influence the unique temporal and spatial association of the deposit styles.

• Proceedings of the KSEEG Conference
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• 2002.10a
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• pp.119-136
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• 2002

Contrasts in the style of the gold-silver mineralization in geologic and tectonic settings in Korea, together with radiometric age data, reflect the genetically different nature of hydrothermal activities, coinciding with the emplacement age and depth of Mesozoic magmatic activities. It represents a clear distinction between the plutonic settings of the Jurassic Daebo orogeny and the subvolcanic environments of the Cretaceous Bulgugsa igneous activities. Dunng the Daebo igneous activities (c.a. 200～150 (\ulcorner) Ma) coincident with orogenic time, gold mineralization took place between c.a. 195 and 135 (127 \ulcorner) Ma. The Jurassic Au deposits commonly show several characteristics; prominent association with pegmatites, low Ag/Au ratios In the ore-concentrating parts, massive vein morphology and a distinctively simple mineralogy including Fe-rich sphalerite, galena, chalcopyrite, arsenopyrite, Au-rich electrum, pyrrhotite and/or pyrite. During the Bulgugsa igneous activities (110～50 Ma), the precious-metal deposits are generally characterized by such features as complex vein morphology, medium to high Ag/Au ratios in the ore concentrates, and diversity of ore minerals including base-metal sulfides, pyrite, arsenopyrite, Ag-rich eletrum and native silver with Ag sulfides, Ag-Sb-As sulfosalts and he tellurides. Vein morphology, mineralogical, fluid inclusion and stable isotope results indicate the diverse genetic natures of hydrothermal systems in Korea. The Jurassic Au-dominant deposits (orogenic type) were formed at the relatively high temperature (about 300$^{\circ}$ to 45$0^{\circ}C$) and deep-crustal level (4.0$\pm$1.5 kb) from the hydrothermal fluids containing more amounts of magmatic waters ($\delta$$^{18}$ $O_{H2O}$; 5～10$\textperthousand$). It can be explained by the dominant ore-depositing mechanisms as $CO_2$ boiling and sulfidation, suggestive of hypo- to mesothermal environments. In contrast, the Cretaceous Au-dominant (l13～68 Ma), Au-Ag (108～47 Ma) and AE-dominant (103～45 Ma) deposits, which correspond to volcanic-plutonic-related type, occurred at relatively low temperature (about 200$^{\circ}$ to 35$0^{\circ}C$) and shallow-crustal level (1.0$\pm$0.5 kb) from the ore-forming fluids containing more amounts of less-evolved meteonc waters ($\delta$$^{18}$ $O_{H2O}$;-10～5$\textperthousand$). These characteristics of the Cretaceous precious-metal deposits can be attributed to the complekities in the ore-precipitating mechanisms (mixing, boiling, cooling), suggestive of epi- to mesothermal environments. Therefore, the differences of the emplacement depth between the Daebo and the Bulgugsa igneous activities directly influence the unique temporal and spatial association of the deposit styles.les.

• Archives of Pharmacal Research
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• v.23 no.2
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• pp.187-195
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• 2000

Nucleotide sequence extending 2,3-dihydroxybiphenyl 1,2-dioxygenase gene (pcbC) and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase gene (pcbD) of Pseudomonas sp. DJ-12 was previously analyzed and the two genes were present in the order of pcbD-pcbC preceded by a promoter from Pseudomonas sp. DJ-12. In this study, a 3.8-kb nucleotide sequence located downstream of the pcbC gene was analyzed to have three open reading frames (ORFs) that are designated as orf1, pcbE and orf2 genes. All of the ORFs were preceded by each ribosome-binding sequence of 5-GGAXA-3 (X=G or A). However, no promoter-like sequence and transcription terminator sequence were found in the analyzed region, downstream of pcbC gene. Therefore, the gene cluster appeared to be present in the order of pcbD-pcbC-orf1-pcbE-orf2 as an operon, which is unique organization characterized so far in biphenyl- and PCB-degrading bacteria. The orf1 gene was composed of 1,224 base pairs which can encode a polypeptide of molecular weight 44,950 containing 405 amino acid residues. A deduced amino acid sequence of the orf1 gene product exhibited 21-33％ identity with those of indole dioxygenase and phenol hydroxylase components. The pcbE gene was composed of 783 base pairs encoding 2-hydroxypenta-2,4-dienoate hydratase involved in the 4-chlorobiphenyl catabolism. The orf2 gene was composed of 1,017 base pairs encoding a polypeptide of molecular weight 37,378 containing 338 amino acid residues. A deduced amino acid sequence of the orf2 gene product exhibited 31％ identity with that of a nitrilotriacetate monooxygenase component.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.5
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• pp.393-399
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• 2004

Useful genes can be Screened from various environments by construction of metagenomic DNA libraries. In this study, water samples were collected from several lakes in mid Korea, and analyzed by T-RFLP to examine diversities of the microbial communities. The crude DNAs r were extracted by the SDS-based freezing-thawing method, and then further purified using an $UltraClean^{TM}$ kit (MoBio, USA). The metagenomic libraries were constructed with the DNAs partially digested with EcoR I, BamH I, and Sac II in Escherichia coli DH 10B using the pBACe3.6 vector. About 44.0 Mb of metagenomic libraries were obtained with average inserts 13-15 kb in size. The bphC genes responsible for degradation of aromatic hydrocarbons via mets-cleavage were identified from the metagenomic libraries by colony hybridization using the bphC specific sequence as a probe. The 2,3-dihydroxybiphenyl (2, 3-DHBP) dioxygenase gene (bphC ), capable of degradation of 2,3-DHBP, was cloned and its nucleotide Sequences analyzed. The genes consisted of 966 and 897 base pairs with an ATG initiation codon and a TGA termination codon. The activity of the 2,3-DHBP dioxygenase was highly expressed to 2,3-DHBP and Showed a broad substrate range to 2,3-DHBP, catechol, 3-methylcatechol and 4-methylcatechol. These results in-dicated that the bphC gene identified from the metagenomes derived from lake water might be useful in the development of a potent strain for degradation of aromatic pollutants.

• The Plant Pathology Journal
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• v.25 no.3
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• pp.205-212
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• 2009

Colletotrichum acutatum was the main cause of the recent outbreaks of anthracnose on pepper fruit in Korea. To facilitate molecular analysis of C. acutatum, we generated an arginine auxotrophic mutant of the C acutatum strain JC24 using a targeted gene replacement strategy. A 3.3-kb genomic region carrying an ortholog (designated CaARG2) of the fungal gene encoding N-acetylglutamate synthase, the first enzyme of arginine biosynthesis in fungi, was deleted from the fungal genome. The mutant exhibited normal growth only when arginine was exogenously supplied into the culture medium. Transformation of the arginine auxotrophic mutant with a plasmid DNA carrying an intact copy of CaARG2, which was smaller than the deleted region in the mutant, not only caused random vector insertions in the fungal genome, but also recovered both hyphal growth and pathogenicity of the mutant to the wild-type level. Using this new selection system, we have successfully developed a restriction enzyme-mediated integration procedure, which would provide an economically efficient random mutagenesis method in C. acutatum.

• Proceedings of the Korea Society of Poultry Science Conference
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• 1999.11a
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• pp.34-50
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• 1999

A cDNA encoding chicken interferon-gamma (chIFN-${\gamma}$) was amplified from P34, a CD4$^{+}$ T-cell hybridoma by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into pUC18. THe sequences of cloned PCR products were determined to confirm the correct cloning. Using this cDNA as probe, chicken genomic library from White Leghorn spleen was screened. Phage clones harboring chicken interferon-gamma (chIFN-${\gamma}$) were isolated and their genomic structure elucidated. The chIFN-${\gamma}$ contains 4 exons and 3 introns spanning over 14 kb, and follows the GT/AG rule for correct splicing at the exon/intron boundaries. The four exons encode 41, 26, 57 and 40 amino acids, respectively, suggesting that the overall structure of IFN-${\gamma}$ is evolutionairly conserved in mammalian and avian species. The 5’-untranslated region and signal sequences are located in exon 1. Several AT-rich sequences located in the fourth exon may indicate a role in mRNA turnover. The 5’-flanking region contains sequences homologous to the potential binding sites for the mammalian transcription factors, activator protein-1(AP-1) activator protein-2(AP-2) cAMP-response element binding protein(CREB), activating transcription factor(ATF), GATA-binding fator(GATA), upstream stimulating factor(USF), This suggests that the mechanisms underlying transcriptional regulation of chicken and mammalian IFN-${\gamma}$ genes may be similar.r.

• Radiation Oncology Journal
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• v.14 no.4
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• pp.265-279
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• 1996

Damage produced by radiation elicits a complex response in mammalian cells, including growth rate changes and the induction of a variety of genes associated with growth control and apoptosis. At doses of 10,000 cGy or greater, the exposed individual was killed in a matter of minutes to a couple of days, with symptoms consistent with pathology of the central nervous system(CNS) including degenerative changes. The nature of the damage in irradiated cells underlies the unique hazards of ionizing radiation. Radiation injury to CNS is a rare event in clinical medicine, but it is catastrophic for the patient in whom it occurs. The incidence of cerebral necrosis has been reported as high as 16% for doses greater than 6,000 cGy. In this study, the effect of radiation on brain tissue was studied in vivo. Jun and p53 genes in the rat brain were induced by whole body irradiation of rat with 600Co in doses between 1 Gy and 100 Gy and analyzed for expression of jun and p53 genes at the postirradiation time up to 6 hours. Northern analyses were done using 1.8 Kb & 0.8 Kb-pGEM-2-JUN/Eco RI/Pst I fragments, 2.0 Kb-php53B/Bam HI fragment and ,1.1 Kb-pBluescript SK--ACTIN/Eco RI fragment as the digoxigenin or [${\alpha}^{32}P$] dCTPlabeled probes for Jun, p53 and ${\beta}$-actin genes, respectively. Jun gene seemed to be expressed near the threshold levels in 1 hour after irradiation of $^{60}$Co in dose less than 1 Gy and was expressed in maximum at 1 hour after irradiation of $^{60}$Co in dose of 30 Gy. Jun was expressed increasingly with time until 5 or 6 hours after irradiation of $^{60}$Co in doses of 1 Gy and 10 Gy. After irradiation of $^{60}$Co in dose between 20 Gr and 100 Gy, the expression of Jun was however increased to peak in 2 hours and decreased thereafter. p53 gene in this study also seemed to be expressed near the threshold levels in 1 hour after irradiation of $^{60}$Co in dose less than 1 Gy and was expressed in maximum at 6 hours after irradiation of $^{60}$Co in dose of 1 Gy, p53 was expressed increasingly with time until 5 or 6 hours after irradiation of $^{60}$Co in dose between 1 Gy and 40 Gy. After irradiation of $^{60}$Co in doses of 50 Gy and 100 Gy, the expression of p53 was however increased to peak in 2 hours and decreased thereafter. The expression of Jun and p53 genes was not correlative in the brain tissue from rats. It seemed to be very important for the establishment of the optimum conditions for the animal studies relevant to the responses of genes inducible on DNA damage to ionizing radiation in mammalian cells. But there are many limitations to the animal studies such as the ununiform patterns of gene expression from the tissue because of its complex compositions. It is necessary to overcome the limitations for development of in situ Northern analysis.