• Microbiology and Biotechnology Letters
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• v.16 no.5
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• pp.343-347
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• 1988

The promoters of alkali-tolerant Bacillus sp. had been cloned in the promoter probe vector pPL703 and recombinant plasmid p-12 had been constructed. As a result of subcloning, two different promoters were found to exist in the cloned 2.9 kb promoter fragment and two recombinant plasmids p-l2B1 and p-l2B2, each harboring different promoter, were constructed. The promoter activity, which was expressed in the CAT specific activity, of p-l2B1 was 7 times higher than that of p-l2B2. The promoter activity as a function of growth revealed that both promoters of p-l2B1 and p-l2B2 were expressed after the late logarithmic growth phase and repressed in the presence of 1.0％ (w/v) glucose.

• Molecules and Cells
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• v.27 no.5
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• pp.583-589
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• 2009

GTP cyclohydrolase I (GTPCH) is a key enzyme in the de novo synthesis of tetrahydrobiopterin. Previously, the Drosophila melanogaster GTPCH gene has been shown to be expressed from two different promoters (P1 and P2). In our study, the 5'-flanking DNA regions required for P1 and P2 promoter activities were characterized using transient expression assay. The DNA regions between -98 and +31, and between -73 and +35 are required for efficient P1 and P2 promoter activities, respectively. The regions between -98 and -56 and between -73 and -41 may contain critical elements required for the expression of GTPCH in Drosophila. By aligning the nucleotide sequences in the P1 and P2 promoter regions of the Drosophila melanogaster and Drosophila virilrs GTPCH genes, several conserved elements including palindromic sequences in the regions critical for P1 and P2 promoter activities were identified. Western blot analysis of transgenic flies transformed using P1 or P2 promoter-lacZ fusion plasmids further revealed that P1 promoter expression is restricted to the late pupae and adult developmental stages but that the P2 promoter driven expression of GTPCH is constitutive throughout fly development. In addition, X-gal staining of the embryos and imaginal discs of transgenic flies suggests that the P2 promoter is active from stage 13 of embryo and is generally active in most regions of the imaginal discs at the larval stages.

• Journal of Life Science
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• v.27 no.12
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• pp.1403-1409
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• 2017

In this study, the xylitol dehydrogenase (XYL2) gene was expressed in Saccharomyces cerevisiae as a host cell for ease of use in the degradation of lignocellulosic biomass (xylose). To select suitable expression systems for the S.XYL2 gene from S. cerevisiae and the P.XYL2 gene from Pichia stipitis, $pGMF{\alpha}-S.XYL2$, $pGMF{\alpha}-P.XYL2$, $pAMF{\alpha}-S.XYL2$ and $pAMF{\alpha}-P.XYL2$ plasmids with the GAL10 promoter and ADH1 promoter, respectively, were constructed. The mating factor ${\alpha}$ ($MF{\alpha}$) signal sequence was also connected to each promoter to allow secretion. Each plasmid was transformed into S. cerevisiae $SEY2102{\Delta}trp1$ strain and the xylitol dehydrogenase activity was investigated. The GAL10 promoter proved more suitable than the ADH1 promoter for expression of the XYL2 gene, and the xylitol dehydrogenase activity from P. stipitis was twice that from S. cerevisiae. The xylitol dehydrogenase showed $NAD^+$-dependent activity and about 77% of the recombinant xylitol dehydrogenase was secreted into the periplasmic space of the $SEY2102{\Delta}trp1/pGMF{\alpha}-P.XYL2$ strain. The xylitol dehydrogenase activity was increased by up to 41% when a glucose/xylose mixture was supplied as a carbon source, rather than glucose alone. The expression system and culture conditions optimized in this study resulted in large amounts of xylitol dehydrogenase using S. cerevisiae as the host strain, indicating the potential of this expression system for use in bioethanol production and industrial applications.

• Tuberculosis and Respiratory Diseases
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• v.73 no.1
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• pp.11-21
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• 2012

Background: While qualitative analysis of methylation has been reviewed, the quantitative analysis of methylation has rarely been studied. We evaluated the methylation status of CDKN2A, $RAR{\beta}$, and RASSF1A promoter regions in non-small cell lung carcinomas (NSCLCs) by using pyrosequencing. Then, we evaluated the association between methylation at the promoter regions of these tumor suppressor genes and the clinicopathological parameters of the NSCLCs. Methods: We collected tumor tissues from a total of 53 patients with NSCLCs and analyzed the methylation level of the CDKN2A, $RAR{\beta}$, and RASSF1A promoter regions by using pyrosequencing. In addition, we investigated the correlation between the hypermethylation of CDKN2A and the loss of $p16^{INK4A}$ immunoexpression. Results: Hypermethylation of CDKN2A, $RAR{\beta}$, and RASSF1A promoter regions were 16 (30.2%), 22 (41.5%), and 21 tumors (39.6%), respectively. The incidence of hypermethylation at the CDKN2A promoter in the tumors was higher in undifferentiated large cell carcinomas than in other subtypes (p=0.002). Hyperrmethylation of CDKN2A was significantly associated with $p16^{INK4A}$ immunoexpression loss (p=0.045). With regard to the clinicopathological characteristics of NSCLC, certain histopathological subtypes were found to be strongly associated with the loss of $p16^{INK4A}$ immunoexpression (p=0.016). Squamous cell carcinoma and undifferentiated large cell carcinoma showed $p16^{INK4A}$ immunoexpression loss more frequently. The Kaplan-Meier survival curves analysis showed that methylation level and patient survival were barely related to one another. Conclusion: We quantitatively analyzed the promoter methylation status by using pyrosequencing. We showed a significant correlation between CDKN2A hypermethylation and $p16^{INK4A}$ immunoexpression loss.

• Microbiology and Biotechnology Letters
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• v.16 no.2
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• pp.126-130
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• 1988

Promoters of an alkali-tolerant Bacillus sp. isolated from soil have been cloned in Bacillus subtilis using promoter probe vector pPL703. The CAT specific activity of a clone harboring the strongest promoter activity among these transformants was 8.01. This activity was 2.5 times higher than that of Bacillus subtilis harboring expression vector pPL708 and was increased after the end of the logarithmic growth phase. In the 2.8kb of inserted DNA fragment, BamHI and Sal I recognition sites were located.

• Journal of Plant Biology
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• v.35 no.2
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• pp.155-163
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• 1992

To elucidate the regulatory mechanism of virE operon in Agrobacterium tumefaciens pTiA6 plasmid at the molecular level, the regulatory site of virE promoter was determined using truncated virE recombinant plasmids obtained by 5' deletion analysis of virE promoter. The size of deleted nucleotides of p]S201, a functional recombinant plasmid, was found to be about 130 nucleotides from 5'-end of virE promoter. On the other hand the size of deleted nucleotides of p]S301, nonfunctional recombinant plasmid, was identified 263 nucleotides by DNA sequencing. Hence it was thought that the essential site of virE promoter was located between about 130th nucleotide and 263th nucleotide. Since the inverted repeat sequence (AACTTTGCGCTATAGGCAMGTT) is included in this essential site of virE promoter, it could be the first recognition site of the RNA polymerase in virE promoter.omoter.

• Microbiology and Biotechnology Letters
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• v.23 no.3
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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.

• Journal of Microbiology and Biotechnology
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• v.31 no.6
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• pp.833-839
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• 2021

Bacillus subtilis CH3-5 isolated from cheonggukjang secretes a 28 kDa protease with a strong fibrinolytic activity. Its gene, aprE3-5, was cloned and expressed in a heterologous host (Jeong et al., 2007). In this study, the promoter of aprE3-5 was replaced with other stronger promoters (P_cry3A, P₁₀, P_SG1, P_srfA) of Bacillus spp. using PCR. The constructed chimeric genes were cloned into pHY300PLK vector, and then introduced into B. subtilis WB600. The P10 promoter conferred the highest fibrinolytic activity, i.e., 1.7-fold higher than that conferred by the original promoter. Overproduction of the 28 kDa protease was confirmed using SDS-PAGE and fibrin zymography. RT-qPCR analysis showed that aprE3-5 expression was 2.0-fold higher with the P10 promoter than with the original promoter. Change of the initiation codon from GTG to ATG further increased the fibrinolytic activity. The highest aprE3-5 expression was observed when two copies of the P₁₀ promoter were placed in tandem upstream of the ATG initiation codon. The construct with P10 promoter and ATG and the construct with two copies of P10 promoter in tandem and ATG exhibited 117% and 148% higher fibrinolytic activity, respectively, than that exhibited by the construct containing P10 promoter and GTG. These results confirmed that significant overproduction of a fibrinolytic enzyme can be achieved by suitable promoter modification, and this approach may have applications in the industrial production of AprE3-5 and related fibrinolytic enzymes.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.762-766
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• 2001

The nar promoter of Escherichia coli was known to induce maximally under anaerobic or microaerobic conditions in the presence of nitrate. In this study, the nar promoter was tested to see whether the expression level of a reporter gene which fused lacZ gene at nar promoter's downstream, in the some gram negative host strains(Agrobacterium, Pseudomonas and Rhizobium). A nar promoter system(Combination of nar promoter and gram negative strain) was grown under aerobic conditions to absorbance at 600 nm of nearly 2.0 and then, the nar promoter was induced by lowering DO to 1-2% with alternating microaerobic and aerobic condition in the fermentor cultures, using different gram negative hosts. For a wild type nar promoter (pNW61), it was possible to maintain production of ${\beta}-galactosidase$ activity per cell(specific ${\beta}-galactosidase$ activity) at 14,000, 9600, 45 Miller units in the presence of 1% nitrate. and for a nitrate - independent nar promoter (pNW618) at 12,000, 10,400 and 58 Miller units in the absence of nitrate ion, respectively.

• Journal of Microbiology and Biotechnology
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• v.3 no.3
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• pp.146-155
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• 1993

A DNA fragment from an alkali-tolerent Bacillus sp., conferring strong promoter activity, was subcloned into the promoter probe plasmid pPL703 and the nucleotide sequence of this promoter region was determined. The sequence analysis suggested that this highly efficient promoter region containing the complex clustered promoters comprised three kinds of promoters (P1, P2 and P3), which are transcribed by $\sigma^B (formerly \sigma^{37}), \sigma^E(formerly \sigma^{29}) and \sigma^A (formerly \sigma^{43})$ RNA polymerase holoenzymes which play major rules at the onset of endospore formation, during sporulation and at the vegetative phase of growth, respectively. S1 nuclease mapping experiments showed that all three promoters had staggered transcription initiation points. The results of chloramphenicol acetyltransferase assay after the subcloning experiments also indicated that the expression of these clustered promoters was correlated with the programs of growth and endospore development. Promoter P1, P2 and P3 were preceded by 75% AT, 79% AT and 81% AT regions, respectively, and a partial deletion of AT-rich region prevented transcription from promoter P1 in vivo. Two sets of 5 -AGTGTT-3 sequences and inverted repeat sequences located around the promoter P1 were speculated as the possible cis acting sites for the catabolite repression in B. subtilis. In vivo transcripts from these sequence regions may be able to form a secondary structure, however, the possibility that a regulatory protein induced by the excess amount of glucose could be bound to such a domain for crucial action remains to be determined.