• Food Science and Biotechnology
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• v.17 no.6
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• pp.1221-1227
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• 2008

The application of the cellulase gene (celA) as a selection marker of food-grade integration system was investigated in Lactobacillus (Lb.) casei, Lactococcus lactis, and Leuconostoc (Leu.) mesenteroides. The 6.0-kb vector pOC13 containing celA from Clostridium thermocellum with an integrase gene and a phage attachment site originating from bacteriophage A2 was used for site-specific recombination into chromosomal DNA of lactic acid bacteria (LAB). pOC13 was also equipped with a broad host range plus replication origin from the lactococcal plasmid pWV01, and a controllable promoter of nisA ($P_{nisA}$) for the production of foreign proteins. pOC13 was integrated successfully into Lb. casei EM116, and pOC13 integrants were easily detectable by the formation of halo zone on plates containing cellulose. Recombinant Lb. casei EM 116::pOC13 maintained these traits in the absence of selection pressure during 100 generations. pOC13 was integrated into the chromosome of L. lactis and Leu. mesenteroides, and celA acted as an efficient selection marker. These results show that celA can be used as a food-grade selection marker, and that the new integrative vector could be used for the production of foreign proteins in LAB.

• Korean Journal of Animal Reproduction
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• v.25 no.4
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• pp.305-315
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• 2001

In this study, the production of transgenic embryo was attempted by microinjection or round spermatid cultured with foreign DNA. At first, the expression of haploid spermatids specific gene, mTP1 in mouse and hPrm2 in hamster spermatids were investigated by RT-PCR method in testes of young mice and hamster testis. The specific gene expression first appeared at 18 days post partum (dpp) in mice spermatid and 20 dpp in hamster spermatid. Therefore, the round spermatids isolated from 17 dpp mice and 19 dpp hamster were used for the introduction of foreign EGFP gene into haploid round spermatids. For the introduction of EGFP gene haploid round spermatids suspended in medium including EGFP gene were treated with a different electric field strength at 0.11, 0.18 and 0.44 ㎸/cm. After electrical stimulation, viability of testicular sperm cells and 67.6%, 66.4% and 49.9%, in mice and 62.6%, 57.9% and 27% in hamster, respectively. These values were significantly lower than those of non-treated control groups 80.5% in mouse and 69.1% in hamster After 72 hrs culture, the highest expression rate of EGFP gene, 28.5% in mice and 32.1% in hamster were obtained from tile spermatogenic cells electroporated by the field strength or 0.18 ㎸/cm. Then, the ability of fertilization and embryonic development of haploid spermatids transfected with foreign EGFP gene were estimated by the microinjection of spermatids into hamster oocytes. The Irate pronuclear formation rate (77.5%) was lower than non-treated control (80%), and the cleavage rate of the treated group (58.8%) was lower than control (65%). To prove the foreign EGFP integration in hamster embryos, 2-cell stage hamster embryos were subjected to the observation under the fluorescence microscope, and the PCR analysis. As a result, about 44% of 2-cell embryos were showed the integration of EFGP gene into their genome. Therefore, These results suggest the possibility to produce transgenic hamsters by microinjection of haploid spermatid transfected with foreign DNA.

• Journal of Life Science
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• v.24 no.10
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• pp.1046-1054
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• 2014

Beta-1,3-glucanase is widely used in various biotechnological and industrial processes, with over-production required to enable versatile utilization. We examined the overexpression of ${\beta}$-1,3-glucanase (EXGA) from Aspergillus oryzae using ${\delta}$-sequence-mediated integration. We constructed $pRS{\delta}$-exgA and $pRS{\delta}K$-exgA plasmids for integration of the EXGA gene into various chromosomes of Saccharomyces cerevisiae. These plasmids contain the ADH1 promoter for constitutive expression, a signal sequence (exoinulinase signal sequence [INU1 s.s]) for secretory production, and a ${\delta}$-sequence for integration of ${\beta}$-1,3-glucanase. The $pRS{\delta}$-exgA plasmid was transformed into the S. cerevisiae $BY4742{\Delta}exg1$ strain, and ${\beta}$-1.3-glucanase was stably overexpressed and secreted. Another plasmid, $pRS{\delta}K$-exgA, was introduced into the S. cerevisiae $BY4742{\Delta}exg1$ (YKY082) strain, and overexpression of ${\beta}$-1,3-glucanase was examined by inducible integration under geneticin selection. The activity of ${\beta}$-1,3-glucanase increased in accordance with a rise in the geneticin concentration, with 0.8 mg/ml of geneticin suitable for overexpression of ${\beta}$-1,3-glucanase. Subsequently, $pRS{\delta}K$-exgA was repeatedly transformed for sequential ${\delta}$-integration. The activity of ${\beta}$-1,3-glucanase reached about 0.063 unit/ml/$OD_{600}$, 0.095 unit/ml/$OD_{600}$, 0.131 unit/ml/$OD_{600}$ and 0.165 unit/ml/$OD_{600}$ by the first, second, third, and fourth round of integration, respectively. According to the increase in the activity of ${\beta}$-1,3-glucanase by sequential ${\delta}$-integration, the copy number (integration rate) of the EXGA gene also increased in various chromosomes. These results suggest that recombinant ${\beta}$-1,3-glucanase activity can be sequentially increased by repeated ${\delta}$-sequence integration.

• Korean Journal of Microbiology
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• v.31 no.2
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• pp.136-140
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• 1993

The A. nidulans expression vector which contained trpC marker gene from A. nidulans was constructed to produce glucoamy]ase. The recombinant plasmid was introduced into auxotrophic mutant A. nidulans B17. Southern blot analysis of the genomic DNA from transformant showed that pKHG2 DNA had integrated into the A. nidulans chromosomes. Northern analysis of the total RNA from transform ant showed that mRNA of glucoamylase gene was synthesized in induction condition. Specific activity of glucoamylase was increased in transform ants. G]ucoamylase was shown to be active in non-denaturing acrylamide gel.

• Applied Biological Chemistry
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• v.28 no.1
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• pp.36-47
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• 1985

$HIS_5$ gene of Saccaromyces cerevisiae was cloned into pBR 322 and also into pSH 610 shuttle vector. $HIS_5$ gene was expressed as promoter of lactose operon(lacZ). And $HIS_5-lacZ$ fusion was intergrated into chromosome III of yeast. $HIS_5$ gene in yeast growth was controlled general amino acid control mechanism.

• Journal of Microbiology and Biotechnology
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• v.26 no.4
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• pp.725-729
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• 2016

A novel genome-engineering tool in Clostridium acetobutylicum was developed based on single-crossover homologous recombination. A small-sized non-replicable plasmid, pHKO1, was designed for efficient integration into the C. acetobutylicum genome. The integrated pHKO1 plasmid backbone, which included an antibiotic resistance gene, can be excised in vivo by Flp recombinase, leaving a single flippase recognition target sequence in the middle of the targeted gene. Since the pSHL-FLP plasmid, the carrier of the Flp recombinase gene, employed the segregationally unstable pAMβ1 replicon, the plasmid was rapidly cured from the mutant C. acetobutylicum. Consequently, our method makes it easier to engineer C. acetobutylicum.

• Toxicological Research
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• v.31 no.1
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• pp.1-9
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• 2015

Environmental toxicants such as toxic metals can alter epigenetic regulatory features such as DNA methylation, histone modification, and non-coding RNA expression. Heavy metals influence gene expression by epigenetic mechanisms and by directly binding to various metal response elements in the target gene promoters. Given the role of epigenetic alterations in regulating genes, there is potential for the integration of toxic metal-induced epigenetic alterations as informative factors in the risk assessment process. Here, we focus on recent advances in understanding epigenetic changes, gene expression, and biological effects induced by toxic metals.

• KSBB Journal
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• v.24 no.3
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• pp.279-286
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• 2009

Streptomyces is well known for their ability to synthesize enormous varieties of antibiotics as secondary metabolites. Among them, S. avermitilis produces avermectins, a group of antiparasitic agents used in human and veterinary medicine. However, S. avermitilis also produces oligomycin, which is a potential toxic inhibitor of oxidative phosphorylation in mammalian cells. Therefore, we decided to disrupt oligomycin synthetase gene to prevent co-production of oligomycin in S. avermitilis. To create plasmid for disruption, the smallest gene of oligomycin synthetase gene cluster was obtained by PCR from S. avermitilis chromosome. Then, apramycin resistance gene was inserted in oligomycin synthetase gene for selection. After transformation of this plasmid, oligomycin synthetase gene (olmA5) in the chromosome was displaced with disruption cassette on the plasmid via homologous recombination. As a result of this gene replacement, we obtained mutants (olmA5::apra) that no longer makes the toxic oligomycin. And the mutants confirmed by PCR and HPLC analysis. However, showed no increasement of avermectin production in the mutant was observed.

• Molecules and Cells
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• v.38 no.11
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• pp.950-958
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• 2015

BCS1L gene encodes mitochondrial protein and is a member of conserved AAA protein family. This gene is involved in the incorporation of Rieske FeS and Qcr10p into complex III of respiratory chain. In our previous study, AluYRa2-derived alternative transcript in rhesus monkey genome was identified. However, this transcript has not been reported in human genome. In present study, we conducted evolutionary analysis of AluYRa2-exonized transcript with various primate genomic DNAs and cDNAs from humans, rhesus monkeys, and crabeating monkeys. Remarkably, our results show that AluYRa2 element has only been integrated into genomes of Macaca species. This Macaca lineage-specific integration of AluYRa2 element led to exonization event in the first intron region of BCS1L gene by producing a conserved 3' splice site. Intriguingly, in rhesus and crabeating monkeys, more diverse transcript variants by alternative splicing (AS) events, including exon skipping and different 5' splice sites from humans, were identified. Alignment of amino acid sequences revealed that AluYRa2-exonized transcript has short N-terminal peptides. Therefore, AS events play a major role in the generation of various transcripts and proteins during primate evolution. In particular, lineage-specific integration of Alu elements and species-specific Alu-derived exonization events could be important sources of gene diversification in primates.

• Journal of Microbiology and Biotechnology
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• v.27 no.2
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• pp.306-315
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• 2017

Metabolic engineering with a high-yielding mutant, A. terreus AN37, was performed to enhance the production of itaconic acid (IA). Reportedly, the gene cluster for IA biosynthesis is composed of four genes: reg (regulator), mtt (mitochondrial transporter), cad (cis-aconitate decarboxylase), and mfs (membrane transporter). By overexpressing each gene of the IA gene cluster in A. terreus AN37 transformed by the restriction enzyme-mediated integration method, several transformants showing high productivity of IA were successfully obtained. One of the AN37/cad transformants could produce a very high amount of IA (75 g/l) in shake-flask cultivations, showing an average of 5% higher IA titer compared with the high-yielding control strain. Notably, in the case of the mfs transformants, a maximal increase of 18.3% in IA production was observed relative to the control strain under the identical fermentation conditions. Meanwhile, the overexpression of reg and mtt genes showed no significant improvements in IA production. In summary, the overexpressed cis-aconitate decarboxylase (CAD) and putative membrane transporter (MFS) appeared to have positive influences on the enhanced IA productivity of the respective transformant. The maximal increases of 13.6~18.3% in IA productivity of the transformed strains should be noted, since the parallel mother strain used in this study is indeed a very high-performance mutant that has been obtained through intensive rational screening programs in our laboratory.