• 생명과학회지
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• 제14권3호
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• pp.429-434
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• 2004

Zymomonas mobilis 유래 levansucrase 유전자(levU)를 GAL1 promoter 하류에 연결시킨 pYES-levU와 GAL10 promoter 하류에 Kluyveromyces marxianus exoinulinase의 분비 신호서열(INU1 ss) 하류에 연결시킨 pYInu-levU를 각각 구축하였다. 이들 plasmid를 invertase 결손 변이주(suc2-$\Delta$9)인 S. cerevisiae SEY2102에 형질전환시켜 고활성 형질전환주를 선발하였다. 효모 형질전환주를 galactose 함유 배지로 배양한 결과, pYES-levU 함유 형질전환주인 경우 levansucrase의 총활성은 7.17U/ml이고, pYInu-levU 함유 형질전환주인 경우 6.61U/ml에 도달하였다. 발현된 levansucrase 약 50% 정도가 배지와 periplasmic space에 존재하였고, INU1 ss에 의한 분비효율 증가는 관찰할 수 없었다. 또한, 효모에서 발현된 재조합 levansucrase는 과당쇄화된 형으로 생산되는 것으로 보여진다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권3호
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• pp.273-276
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• 2006

RNA interference (RNAi), a process of sequence-specific gene suppression, has been known as a natural gene regulatory mechanism in a wide range of lower organisms. Recently, we have reported that a transfection of human U6 promoter (hU6) driven hairpin small-interference RNA (siRNA) plasmid specifically knocks down the target gene by post-transcriptional gene silencing in mammalian cells. Here we report that transfection of polymerase chain reaction (PCR) products, containing human U6 promoter with hairpin siRNA, knocks down the target gene expression in human teratocarcinoma NT-2/D1 cells. Moreover, we showed 3' end termination sequence, 5 Ts, is not critical elements for knocking down in PCR-based siRNA system. Therefore, the PCR-based siRNA system is a promising tool not only for the screening but also to temporally regulate gene expression in the human progenitor cells.

• Molecules and Cells
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• 제23권2호
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• pp.138-144
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• 2007

Previously we showed that the human GM3 synthase gene was expressed during the induction of megakaryocytic differentiation in human leukemia K562 cells by phorbol 12-myristate 13-acetate (PMA). In this study we found that treatment of PMA-induced K562 cells with $G{\ddot{o}}6976$, a specific inhibitor of PKC, and U0126, an inhibitor of the extracellular signal-regulated kinase (ERK) reduced expression of GM3 synthase, whereas wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K) did not. Moreover, activation of ERK and cAMP response element binding protein (CREB) was prevented by pretreatment with $G{\ddot{o}}6976$ and U0126. PMA stimulated the promoter activity of the 5'-flanking region from -177 to -83 region of the GM3 synthase gene, and mutation or deletion of a CREB site located around -143 of the promoter reduced PMA-stimulated promoter activity, as did the inhibitors $G{\ddot{o}}6976$ and U0126. Our results demonstrate that induction of GM3 synthase during megakaryocytic differentiation in PMA-stimulated human leukemia K562 cells depends upon the PKC/ERK/CREB pathway.

• 한국미생물·생명공학회지
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• 제23권3호
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• pp.288-296
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• 1995

To investigate high-level expression of Serratia marcescens metalloprotease (SMP) in Escherichia coli and S. marcescens, we constructed various recombinant plasmids: pSP2, containing SMP gene and lac promoter; pKSP2, containing SMP gene and tac promoter; pTSP2, containing SMP gene, trc99a promoter, and lacI$^{q}$. The recombinant E. coli (pKSP2) strain expressed SMP to a high-level, about 36% of total cellular proteins but accumulated inactive SMP precursors intracellularly, which indicated that E. coli does not have activation and secretion system for SMP. To overproduce active SMP, we transformed S. marcescens with the recombinant plasmids by a modified CaCl$_{2}$ method. The recombinant S. marcescens ATCC27117 (pSP2) containing lac promoter for SMP transcription produced 530 U/ml of active SMP on LB broth, which is about 5.1 times of the SMP yield, 105 U/ml of a control strain, S. marcescens ATCC27117 (pUC19). However, S. marcescens ATCC27117 (pKSP2) containing tac promoter for SMP transcription did not grow healthy and hardly produced SMP. To overcome a harmful effect of the strong tac promoter, we constructed a regulatory plasmid pTSP2 containing a strong trc99a promoter and its repressor gene lacI$^{q}$. When S. marcescens ATCC27117 (pTSP2) was induced with 1.0 mM IPTG after 9 hr cultivation, 2,200 U/ml of SMP was obtained in LB broth, which is about 21 times of that of a control strain.

• 한국미생물·생명공학회지
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• 제32권3호
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• pp.206-211
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• 2004

Pseudomonas aurantiaca 유래 levansucrase 유전자(lscA)를 GAL1 promoter 하류에 연결시킨 pYES-lscA와 CAL10 promoter와 Kluyveromyces marxianus exoinulinase의 분비 신호서열(INU1 ss)하류에 연결시킨 pYInu-lscA를 각각 구축하였다. 이들 plasmid를 invertase 결손 변이주(suc2-$\Delta$9)인 S. cerevisiae SEY2102에 형질전환시켜 고활성 형질전환주를 선발하였다. 효모 형질전환주를 galactose 함유 배지로 배양한 결과, pYES-lscA 함유 형질전환주인 경우 levansucrase의 총활성은 8.62 U/ml이고, pYInu-lscA 함유 형질전환주인 경우 5.43 U/ml에 도달하였다. 발현된 levansucrase의 약 80% 정도가 periplasmic space와 cytopla느에 존재하였고, INU1 ss에 의한 분비효율 증가는 관찰할 수 없었다. 또한, 효모에서 발현된 재조합 levansucrase는 과당쇄화된 형으로 생산되는 것으로 보여진다.

• Genomics & Informatics
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• 제8권4호
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• pp.201-205
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• 2010

The H1 histone family, member N, testis-specific (H1FNT) is exclusively expressed in the testis, and had its possible role for sperm chromatin formation. The purpose of this study is to investigate any genetic association of H1FNT gene with male infertility, especially at the promoter region. We examined the promoter single nucleotide polymorphisms (SNP) of H1FNT gene which is located within transcription factor binding site for its association with male infertility. The statistical analysis showed that the -1129A>T polymorphism was present at a statistically significance in male infertility (p=0.0059 and 0.0349 for hetero and risk type, respectively). The dual-luciferase promoter assay was performed to examine the polymorphic effect of this promoter SNP by the cloning of promoter region (1700bp fragment) into pGL3-basic vector. In our plasmid based reporter system, there is no big difference between wild and risk type. In conclusion, H1FNT -1129A>T promoter SNP is statistically significant with male infertility, especially with subfertile (non-azoospermia) group. Further analysis of its functional polymorphic effect in vivo may provide the biological significance of testis-specific histone with spermatogenesis.

• Molecules and Cells
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• 제24권1호
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• pp.148-151
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• 2007

Porcine endogenous retroviruses (PERVs) in the pig genome represent a potential risk of infection in pig-to-human transplantation and are transmitted vertically. The solitary long terminal repeat (LTR) elements of the PERVs affect the replication properties of the individual viruses via their repeat sequences and by encoding a set of specific transcription factors. We examined the promoter activities of solitary LTR elements belonging to the PERV-A and -B families of the Korean domestic pig (KDP) using luciferase reporters. Three of the LTR structures (of PERV-A5-KDP, PERV-A7-KDP, PERV-A8-KDP) had different promoter activities in human HCT116 cells and monkey Cos7 cells, and potential negatively and positively acting regions affecting transcription were identified by deletion analysis. These data suggest that specific sequences in the U3 region of a given LTR element can affect the activities of promoter or enhancer elements in the PERV.

• Asian-Australasian Journal of Animal Sciences
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• 제31권6호
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• pp.791-797
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• 2018

Objective: Trimethylation of histone 3 (H3) at 4th lysine N-termini (H3K4me3) in gene promoter region was the universal marker of active genes specific to cell lineage. On the contrary, coexistence of trimethylation at 27th lysine (H3K27me3) in the same loci-the bivalent H3K4m3/H3K27me3 was known to suspend the gene transcription in germ cells, and could also be inherited to the developed stem cell. In galline species, throughout example of H3K4m3 and H3K27me3 ChIP-seq analysis was still not provided. We therefore designed and demonstrated such procedures using ChIP-seq and mRNA-seq data of chicken follicular mesenchymal cells and male germ cells. Methods: Analytical workflow was designed and provided in this study. ChIP-seq and RNA-seq datasets of follicular mesenchymal cells and male germ cells were acquired and properly preprocessed. Peak calling by Model-based analysis of ChIP-seq 2 was performed to identify H3K4m3 or H3K27me3 enriched regions ($Fold-change{\geq}2$, $FDR{\leq}0.01$) in gene promoter regions. Integrative genomics viewer was utilized for cellular retinoic acid binding protein 1 (CRABP1), growth differentiation factor 10 (GDF10), and gremlin 1 (GREM1) gene explorations. Results: The acquired results indicated that follicular mesenchymal cells and germ cells shared several unique gene promoter regions enriched with H3K4me3 (5,704 peaks) and also unique regions of bivalent H3K4m3/H3K27me3 shared between all cell types and germ cells (1,909 peaks). Subsequent observation of follicular mesenchyme-specific genes-CRABP1, GDF10, and GREM1 correctly revealed vigorous transcriptions of these genes in follicular mesenchymal cells. As expected, bivalent H3K4m3/H3K27me3 pattern was manifested in gene promoter regions of germ cells, and thus suspended their transcriptions. Conclusion: According the results, an example of chicken H3K4m3/H3K27me3 ChIP-seq data analysis was successfully demonstrated in this study. Hopefully, the provided methodology should hereby be useful for galline ChIP-seq data analysis in the future.

• Molecules and Cells
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• 제21권2호
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• pp.237-243
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• 2006

Brain-derived neurotrophic factor (BDNF) is a neuromodulator of nociceptive responses in the dorsal root ganglia (DRG) and spinal cord. BDNF synthesis increases in response to nerve growth factor (NGF) in trkA-expressing small and medium-sized DRG neurons after inflammation. Previously we demonstrated differential activation of multiple BDNF promoters in the DRG following peripheral nerve injury and inflammation. Using reporter constructs containing individual promoter regions, we investigated the effect of NGF on the multiple BDNF promoters, and the signaling pathway by which NGF activates these promoters in PC12 cells. Although all the promoters were activated 2.4-7.1-fold by NGF treatment, promoter IV gave the greatest induction. The p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, phosphatidylinositol 3-kinase (PI-3K) inhibitor, LY294003, protein kinase A (PKA) inhibitor, H89, and protein kinase C (PKC) inhibitor, chelerythrine, had no effect on activation of promoter IV by NGF. However, activation was completely abolished by the MAPK kinase (MEK) inhibitors, U0126 and PD98059. In addition, these inhibitors blocked NGF-induced phosphorylation of extracellular signal-regulated protein kinase (ERK) 1/2. Taken together, these results suggest that the ERK1/2 pathway activates BDNF promoter IV in response to NGF independently of NGF-activated signaling pathways involving PKA and PKC.

• 한국미생물·생명공학회지
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• 제30권3호
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• pp.206-209
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• 2002

Bacillus stearothermophilus의 CCTase 유전자(cgtS) 대장균과 효모의 shuttle vector로서 항구적 promoter인 adh l promoter를 함유한 pVT103-U(6.9Kb)에 도입하여 재조합 plasmid pVT-CCTS (9.0Kb)을 구축하고 효모 숙주 S. cerevisiae 2805에서 발현시켰다. 재조합 균주의 항구적 발현계인 2805/pv7-CGTS의 최적 발현조건은 YP배지에 dextrose 2%, pH 5.5, 30"C에서 최적 발효조건이었으며, CCTase의 최대 발현량은 48시간 배양시 0.624unit/mL을 나타내었다. B. stearothermophilus의 signal peptide가 재조합 효모에서도 높은 분비효율을 나타내어서 발현된 효소의 87%가 세포 외로 분비 생산되었다.산되었다.