• Reproductive and Developmental Biology
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• v.37 no.4
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• pp.269-279
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• 2013

Low efficiency of somatic cell nuclear transfer (SCNT) is attributed to incomplete reprogramming of transfered nuclei into oocytes. Trichostatin A (TSA), histone deacetylase inhibitor and 5-aza-2'deoxycytidine (5-aza-dC), DNA methylation inhibitor has been used to enhance nuclear reprogramming following SCNT. However, it was not known molecular mechanism by which TSA and 5-aza-dC improve preimplantation embryo and fetal development following SCNT. The present study investigates embryo viability and gene expression of cloned porcine preimplantation embryos in the presence and absence of TSA and 5-aza-dC as compared to embryos produced by parthenogenetic activation. Our results indicated that TSA treatment significantly improved development. However 5-aza-dC did not improve development. Presence of TSA and 5-aza-dC significantly improved total cell number, and also decreased the apoptotic and autophagic index. Three apoptotic-related genes, Bak, Bcl-xL, and Caspase 3 (Casp3), and three autophagic-related genes, ATG6, ATG8, and lysosomal-associated membrane protein 2 (LAMP2), were measured by real time RT-PCR. TSA and 5-aza-dC treatment resulted in high expression of anti-apoptotic gene Bcl-xL and low pro-apoptotic gene Bak expression compared to untreated NT embryos or parthenotes. Furthermore, LC3 protein expression was lower in NT-TSA and NT-5-aza-dC embryos than those of NT and parthenotes. In addition, TSA and 5-aza-dC treated embryos displayed a global acetylated histone H3 at lysine 9 and methylated DNA H3 at lysine 9 profile similar to the parthenogenetic blastocysts. Finally, we determined that several DNA methyltransferase genes Dnmt1, Dnmt3a and Dnmt3b. NT blastocysts showed higher levels Dnmt1 than those of the TSA and 5-aza-dC blastocysts. Dnmt3a is lower in 5-aza-dC than NT, NTTSA and parthenotes. However, Dnmt3b is higher in 5-aza-dC than NT and NTTSA. These results suggest that TSA and 5-aza-dC positively regulates nuclear reprogramming which result in modulation of apoptosis and autophagy related gene expression and then reduce apoptosis and autophagy. In addition, TSA and 5-aza-dC affects the acetylated and methylated status of the H3K9.

• Journal of Microbiology and Biotechnology
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• v.23 no.1
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• pp.64-68
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• 2013

Previous studies have shown that lactic acid bacteria can inhibit inflammatory responses, but the mechanisms are very little known. In this study, transaction and expression of three proinflammatory factors, iNOS, PTGS-2, and IL8, which are closely related to the inflammatory response, were investigated by luciferase reporter assay and RTPCR in HT-29 cells treated by Lactobacillus acidophilus. The results showed that the live L. acidophilus sharply down-regulated the transcription of these three genes. Because there was a NF-${\kappa}B$ binding site located at -265 bp, -225 bp, and -95 bp upstream of the iNOS, PTGS-2, and IL8 promoters, respectively, we further addressed the effects of NF-${\kappa}B$ on transaction of the three promoters by cotransfection. As was expected, NF-${\kappa}Bs$ remarkably upregulated the activity of the reporter gene and, no effect of NF-${\kappa}B$s on IL-8 promoter transaction was found after NF-${\kappa}B$ binding site mutation of the IL8 promoter in HT-29 cells. In conclusion, the live L. acidophilus decreased the transcriptional activity of NF-${\kappa}B$ and, in turn, inhibited the transaction of NF-${\kappa}B$ on the three proinflammatory factors mentioned above.

• Korean Journal of Environmental Biology
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• v.31 no.4
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• pp.288-294
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• 2013

Recently, sustainable production of biofuel using algal biomass is being pursued because of its enormous potential. First and foremost, securing superior strains to develop an efficient production system for algal biodiesel through screening or genetic improvement of microalgae is necessary. The genus of Botryococcus is regarded as one of the superior microalgae for biodiesel production due to its ability to accumulate high amounts of lipids and hydrocarbons. However, its low growth rate is a bottleneck for large-scale production and commercialization. The purpose of this study is to obtain indigenous Botryococcus strains which possess high lipid content and biomass productivity. The Botryococcus sp. was isolated from the Seobu Reservoir in Jeju Island and identified as Botryococcus sudeticus J2 by comparative analysis of 18s rRNA gene and ITS regions. The biomass productivity and lipid content of B. sudeticus J2 were 0.116 g $L^{-1}day^{-1}$ and 40.1% of dry wt., respectively. This was higher than the value of B. braunii UTEX 572, which is widely regarded as a superior strain among Botryococcus species. The relatively high growth rate of B. sudeticus J2 was achieved under a light intensity of 240 ${\mu}mol$ photons $m^{-2}s^{-1}$ with ambient air spargingwhen compared to 120 ${\mu}mol$ photons $m^{-2}s^{-1}$ with 2% $CO_2$ supply. In summary, it is likely that the isolated B. sudeticus J2 can be used for the mass cultivation and biodiesel production.

• Korean Journal of Microbiology
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• v.50 no.3
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• pp.185-190
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• 2014

The aim of this study was to investigate the prevalence of quinolone resistant E. coli from raw bulk milk and to characterize the resistance determinants. In this study, the gyrA, gyrB, parC, and parE quinolone resistance determining regions (QRDR) were sequenced from quinolone resistant E. coli isolates. Also, the presence of plasmid-mediated quinolone resistance (PMQR) and the expression of efflux pump genes based on quantitative real-time PCR (qRT-PCR) were investigated. Of the 487 coliform bacteria, 9 strains showed nalidixic acid resistance, and 6 of the 9 nalidixic acid resistant isolates were also ciprofloxacin resistant. These 9 strains had a single mutation at codon 83 (S83L) in gyrA, 2 of them had double mutations at codon 83 and 87 (S83L and D87N) in gyrA and 3 of the 9 isolates had single mutations at codon 80 (S80I) in parC. None of the 9 isolates harbored PMQR determinants. Compared with wild-type E. coli ATCC 25922, an over-expression of the acrB gene (2.15-5.74 fold), encoding the pump component of the AcrAB-TolC efflux pump was observed in 4 of 6 ciprofloxacin resistant isolates. This study identified the quinolone resistance mechanism of E. coli isolated from raw milk samples in Gyeonggi-do.

• Journal of Microbiology and Biotechnology
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• v.16 no.11
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• pp.1799-1808
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• 2006

A food-grade integration vector based on site-specific recombination was constructed. The 5.7-kb vector, pIMA20, contained an integrase gene and a phage attachment site originating from bacteriophage A2, with the ${\alpha}$-galactosidase gene from Lactobacillus plantarum KCTC 3104 as a selection marker. pIMA20 was also equipped with a controllable promoter of nisA ($P_{nisA}$) and a signal peptide-encoding sequence of usp45 ($SP_{usp45}$) for the production and secretion of foreign proteins. pIMA20 and its derivatives mediated site-specific integration into the attB-like site on the Lactococcus lactis NZ9800 chromosome. The vector-integrated recombinant lactococci were easily detected by the appearance of blue colonies on a medium containing $X-{\alpha}-gal$ and also by their ability to grow on a medium containing melibiose as the sole carbon source. Recombinant lactococci maintained these traits in the absence of selection pressure during 100 generations. The ${\alpha}-amylase$ gene from Bacillus licheniformis, lacking a signal peptide-encoding. sequence, was inserted downstream of $P_{nisA}\;and\;SP_{usp45}$ in pIMA20, and the plasmid was integrated into the L. lactis chromosome. ${\alpha}-Amylase$ was successfully produced and secreted by the recombinant L. lactis, controlled by the addition and concentration of nisin.

• Horticultural Science & Technology
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• v.31 no.1
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• pp.72-79
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• 2013

Inactivation of the gene coding for dihydroflavonol 4-reductase (DFR) is responsible for the color difference between red and yellow onions (Allium cepa L.). Two inactive DFR-A alleles, DFR-$A^{PS}$ and DFR-$A^{DEL}$, were identified in our previous study. A functional marker was developed on the basis of the premature stop codon that inactivated the DFR-$A^{PS}$ allele. A derived cleaved amplified polymorphic sequences (dCAPS) primer was designed to detect the single nucleotide polymorphism, an A/T transition, which produced the premature stop codon. Digested PCR products clearly distinguished the homozygous and heterozygous red $F_2$ individuals. Meanwhile, to develop a molecular marker for detection of the DFR-$A^{DEL}$ allele in which entire DFR-A gene was deleted, genome walking was performed and approximately 3 kb 5' and 3' flanking sequences of the DFR-$A^R$ coding region were obtained. PCR amplification using multiple primers binding to the extended flanking regions showed that more of the extended region of the DFR-A gene was deleted in the DFR-$A^{DEL}$ allele. A dominant simple PCR marker was developed to identify the DFR-$A^{DEL}$ allele using the dissimilar 3' flanking sequences of the DFR-A gene and homologous DFR-B pseudogene. Distribution of the DFR-$A^{PS}$ and DFR-$A^{DEL}$ alleles in yellow onion cultivars bred in Korea and Japan was surveyed using molecular makers developed in this study. Results showed predominant existence of the DFR-$A^{PS}$ allele in yellow onion cultivars.

• ALGAE
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• v.38 no.1
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• pp.1-22
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• 2023

An enormous body of research is focused on finding ways to commercialize carotenoids produced by the unicellular green alga, Haematococcus, often without the benefit of a sound phylogenetic assessment. Evidence of cryptic diversity in the genus means that comparing results of pigment studies may be confounded by the absence of a phylogenetic framework. Moreover, previous work has identified unnamed strains that are likely candidates for species status. We reconstructed the phylogeny of an expanded sampling of Haematococcus isolates utilizing data from nuclear ribosomal markers (18S rRNA gene, 26S rRNA gene, internal transcribed spacer [ITS]-1, 5.8S rRNA gene, and ITS-2) and the rbcL gene. In addition, we gathered morphological, ultrastructural and pigment data from key isolates of Haematococcus. Our expanded data and taxon sampling support the concept of a new species, H. privus, found exclusively in North America. Despite overlap in numerous morphological traits, results indicate that ratios of protoplast length to width and akinete diameter may be useful for discriminating Haematococcus lineages. High growth rate and robust astaxanthin yield indicate that H. rubicundus (SAG 34-1c) is worthy of additional scrutiny as a pigment source. With the description of H. privus, the evidence supports the existence of at least five, species-level lineages in the genus. Our phylogenetic assessment provides the tools to frame future pigment investigations of Haematococcus in an updated evolutionary context. In addition, our investigation highlighted open questions regarding polyploidy and sexuality in Haematococcus which demonstrate that much remains to be discovered about this green flagellate.

• Korean Journal of Agricultural Science
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• v.22 no.2
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• pp.170-179
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• 1995

This study was conducted to investigate the genetic constitution of blood proteins and enzymes in 238 Korean Native cattle reared at Korean Native Cattle Breeding Center, National Livestock Cooperative Federation. The genetic polymorphisms of post-transferrin-2(pTf-2), transferrin(Tf), post-albumin(pAlb), albumin(Alb), ceruloplamin(Cp), amylase-I(Am-I) and hemoglobin(Hb) were analyzed by using PAGE(polyacrylamide gel electrophoresis) and STAGE(starch gel electrophoresis). The genotypes and gene frequencies were estimated at these loci for each blood proteins and enzymes. The results obtained from this study were summarized as follows : 1. The pTf-2 locus were identified to be genetically controlled by codominant alleles designated pTf-2 F and S, and the distribution of genotypes were 46.22, 46.64 and 7.14% for pTf-2 FF, FS and SS types, and the gene frequencies of the pTf-2 F and S allele were 0,695 and 0.305, respectiveley. 2. The Tf locus were found to be controlled by Tf A, D1, D2 and E alleles, and the distributioin of genotypes were 0.84, 13.87, 13.03, 10.92, 22.27, 12.61, 2.94, 15.51, 6.72 and 1.68% for Tf AA, AD1, AD2, AE, D1D1, D1D2, D1E, D2E and EE types, and the gene frequencies of Tf A, D1, D2 and E were 0.197, 0.430, 0.191 and 0.081, respectively. 3. The pAlb locus were observed to be controlled by two alleles, pAlb F and S, and the distribution of genotypes were 42.86, 33.19 and 23.95% for pAlb FF, FS and SS types, and the gene frequencies were 0.595 and 0.405 for Tf F and S allele, respectively. Also the gene frequencies of Alb was 1.000 of Alb A allele. 4. The Cp locus were identified to be controlled by Cp F and S allele, and the distribution of genotypes were 23.11, 34.87 and 42.02% for Cp FF, FS and SS types, and the gene frequencies were 0.405 and 0.595 for Cp F and S allele, respectively. 5. The Am-I locus were observed to be genetically controlled by Am-I B and C allele, and the distribution of genotypes were 51.26, 16.81 and 31.92% for Am-I BB, BC and CC types, and the gene frequencies of Am-I B and C alleles were 0.597 and 0.403, respectively. 6. The Hb locus were found to be controlled by Hb A and B alleles, and the distribution of genotypes were 93.19, 16.39 and 0.42% for Hb AA, AB and BB types, and the gene frequencies of Hb A and B alleles were 0.914 and 0.086, respectively.

• Korean Journal of Microbiology
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• v.27 no.3
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• pp.169-175
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• 1989

To study the role of mutant precore region in expression and secretion of hapatitis B viral core antigen, we have cloned core antigen gene(HBc) with or without precore region in geterologous expression vectors containing SV40 promoter, yeast promoter, and lambda $P_{L}$ promoter. In COS cells transfected with plasmid containing C-gene with precore region, antigens were detected in both cell extract and cultured medium. However, in the cells transfected with plasmids containing C-gene without precore or with mutated precore region by one nucleotide (T) addition at the nucleotide 1,821, HBcAg was detected only in cell extracts. These results support that the mutation by one nucleotide addition shifted the initiation codon of precore region to 53 nucleotides upward and the elongated precore region also played a major role in the secretion of HBcAg in mammalian cells. In the case of yeast and E. coli, HBcAg was detected only in cell extracts in spite of the presence of precore region, which suggest that precore region could not affect HBcAg secretion in these system.

• Development and Reproduction
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• v.25 no.4
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• pp.257-268
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• 2021

Phthalates and their metabolites are well-known endocrine disrupting chemicals. Di-(2-ethylhexyl) phthalate (DEHP) has been widely used in industry and the exposing possibility to adult is high. In this study, DEHP was treated (133 ㎍/L and 1,330 ㎍/L in drinking water) according to the OECD test guideline 443 to mature female mice and their adrenal gland were examined for histological characteristics and steroidogenic gene expression. The wet weight of the adrenal gland was increased in all administrated groups compared to control. The diameter of zona fasciculata (ZF) was increased by DEHP in both outer ZF and inner ZF but there was no difference in morphology of the cells and arrangements into zona between groups. In addition, the arrangement of extracellular matrix was not different between control and DEHP groups. CYP11B1 was mainly localized at ZF and the intensity was not different between groups. DAX1 was localized in zona glomerulosa (ZG) and ZF, and its expression levels were decreased by DEHP administration. Its level was lower in DEHP133 group than DEHP1330 group. On the other hand, CYP17A1 was localized in ZG of DEHP1330 group. These results suggest that chronic low-dose DEHP exposing may modify the microstructure and function of the adrenal cortical cortex.