• 식물조직배양학회지
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• 제22권4호
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• pp.201-205
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• 1995

Nopaline synthase promoter의 upstream element region을 본딴 nos-RP 요소라고 불리워진 합성 oligomer는 nos wild type promotor의 5' end에서부터 - 101까지를 절단한 promoter의 upstream에 삽입하였다. Nos promoter의 활성은 nos promoter와 연결되어 있는 reporter gene인 Chlorarnphenicol과 $\beta$-glucuronidase유전 인자들이 발현되는 현상을 연구함으로써 측정하였다. 형질 전환된 유전인자를 가지고 있는 담배 식물에 대한 분석은 nos minimal promoter의 활성이 합성 nos-RP 요소가 삽입됨으로써 회복될 수 있음을 보여 주었다. 또한 Nos minimal promoter의 upstream에 nos-RP 요소의 삽입은 여러 가지 환경적인 요소들인 auxin, dithiothreitol, salicylic acid 그리고 methyl jasmonate에 의해서 활성이 증가됨을 보여 주었다.

• KSBB Journal
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• 제7권3호
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• pp.155-160
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• 1992

식량으로 쓰이는 식물 단백질은 공통적으로 Isoleucine, Lysine, Methionine, Threonine, Tryptoplan등 5가지 필수아미노산이 결핍되어있다. 본 연구에서는 이러한 필수아미노산을 다량 함유한 단백질을 발현시킬 수 있는 합성유전자를 fekaqo에서 높은 수준으로 발현시키고자 강한 식물 promoter로 알려진 CaMV 35S, CaMV duplicate 35S promoters를 사용하였다. 형질 전환 및 재분화된 식물을 분석한 결과 본 합성 유전자가 식물 nuclear genome 안으로 도입을 안정하여 잘되었고 mRNA수준까지는 유의적인 증가를 보였으나 단백질 수준에서는 유의적 수준의 증가를 관찰할 수 없었다.

• Molecules and Cells
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• 제40권8호
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• pp.577-586
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• 2017

Phytocystatins (PhyCYSs) are plant-specific proteinaceous inhibitors that are implicated in protein turnover and stress responses. Here, we characterized a PhyCYS from Arabidopsis thaliana, which was designated AtCYS5. RT-qPCR analysis showed that the expression of AtCYS5 in germinating seeds was induced by heat stress (HS) and exogenous abscisic acid (ABA) treatment. Analysis of the expression of the ${\beta}-glucuronidase$ reporter gene under the control of the AtCYS5 promoter showed that AtCYS5 expression during seed germination was induced by HS and ABA. Constitutive overexpression of AtCYS5 driven by the cauliflower mosaic virus 35S promoter led to enhanced HS tolerance in transgenic Arabidopsis, which was characterized by higher fresh weight and root length compared to wild-type (WT) and knockout (cys5) plants grown under HS conditions. The HS tolerance of AtCYS5-overexpressing transgenic plants was associated with increased insensitivity to exogenous ABA during both seed germination and post-germination compared to WT and cys5. Although no HS elements were identified in the 5'-flanking region of AtCYS5, canonical ABA-responsive elements (ABREs) were detected. AtCYS5 was upregulated in ABAtreated protoplasts transiently co-expressing this gene and genes encoding bZIP ABRE-binding factors (ABFs and AREB3). In the absence of ABA, ABF1 and ABF3 directly bound to the ABREs in the AtCYS5 promoter, which activated the transcription of this gene in the presence of ABA. These results suggest that an ABA-dependent pathway plays a positive role in the HS-responsive expression of AtCYS5 during seed germination and post-germination growth.

• 약학회지
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• 제46권2호
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• pp.143-148
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• 2002

The human polyomavirus JC virus is the etiologic agent of progressive multifocal leukoencephalopathy (PML). The JC virus early promoter directs cell-specific expression of the viral replication factor large T antigen, thus transcriptional regulation constitutes a major mechanism of glial tropism in PML. Here we found that pentanucleotide sequence immediately upstream of the TATA sequence functions as a cell-specific silencer in the JC virus transcription. In vitro binding studies showed that synthetic oligonucleotides spanning a pentanucleotide sequence, designated "oligo 2", interacts with nuclear proteins from non-glial cells in a cell-specific manner. Furthermore, the sequence preferentially repressed the heterologous thymidine kinase promoter activity in non-glial cells. We also tested whether JC virus transcription is controlled by DNA methylation. Transient transfection of in vitro methylated JC virus promoter abolished transcription in both the glial and non-glial cells. The repression fold was much larger in glial cells than in non-glial cells. Taken together, this finding suggests that glial cell-specific expression of the JC virus is controlled by DNA methylation as well as cell-specific silencers.

• BMB Reports
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• 제34권6호
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• pp.502-508
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• 2001

A cDNA, encoding the human growth hormone (hGH), was synthesized based on the known 191 amino acid sequence. Its codon usage was optimized for a high level expression in Escherichia coli. Unique restriction sites were incorporated throughout the gene to facilitate mutagenesis in further studies. To minimize an initiation translation problem, a 624-bp cassette that contained a ribosome binding site and a start codon were fused to the hGH-coding sequence that was flanked between the EcoRI and HindIII sites. The whole fragment was synthesized by an overlapped extension of eight long synthetic oligonucleotides. The four-short duplexes of DNA, which were first formed by annealing and filling-in with a Klenow fragment, were assembled to form a complete hGH gene. The hGH was cloned and expressed successfully using a pET17b plasmid that contained the T7 promoter. Recombinant hGH yielded as much as 20% of the total cellular proteins. However, the majority of the protein was in the form of insoluble inclusion bodies. N-terminal amino acid sequencing also showed that the hGH produced in E. coli contained formyl-methionine. This study provides a useful model for synthesis of the gene of interest and production of recombinant proteins in E. coli.

• 한국미생물·생명공학회지
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• 제47권3호
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• pp.337-342
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• 2019

We published that bacterial heme was over-produced in a recombinant Corynebacterium glutamicum expressing 5-aminolevulinic acid synthase ($hemA^+$) under control of a constitutive promoter ($P_{180}$) and the heme-producing C. glutamicum had commercial potentials; as an iron feed additive for swine and as a preservative for lactic acid bacteria. To enhance the heme production, the $hemA^+$ gene was expressed under controls of various promoters in the recombinant C. glutamicum. The $hemA^+$ expression by $P_{gapA}$ (a constitutive glycolytic promoter of glyceraldehyde-3-phosphate dehydrogenase) led 75% increase of heme production while the expression by $P_{H36}$ (a constitutive, very strong synthetic promoter) resulted in 50% decrease compared with the control ($hemA^+$ expression by $P_{180}$ constitutive promoter). The $hemA^+$ expression by a late log-phase activating $P_{sod}$ (an oxidative-stress responding promoter of superoxide dismutase) led 50% greater heme production than the control. The $hemA^+$ expression led by a heat-shock responding chaperone promoter ($P_{dnaK}$) resulted in 121% increase of heme production at the optimized heat-shock conditions. The promoter strength and induction phase are discussed based on the results for the heme production at an industrial scale.

• Journal of Microbiology and Biotechnology
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• 제8권1호
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• pp.34-41
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• 1998

A method was developed for the controlled expression of an antimicrobial peptide magainin in Escherichia coli. A series of concatemeric magainin genes was constructed with a gene amplification vector, and fused to the 3'end of malE gene encoding the affinity ligand, E. coli maltose-binding protein (MBP). The construct directed the synthesis of the fusion protein with the magainin polypeptide fused to the C-terminus of MBP. The fusion protein was expressed in a tightly regulatable expression system which was under the control of an invertible promoter. The MBP-fused magainin monomer was expressed efficiently. However, the expression level of the MBP-fused magainin in E. coli decreased with the increasing size of multimers possibly because of the transcription and translation inhibition by the multimeric peptides. After purification using an amylose affinity column, the fusion protein was digested by factor Xa at a specific cleavage site between the monomers. The recombinant magainin had an antimicrobial activity identical to that of synthetic magainin. This experiment shows that a biologically active, antimicrobial peptide magainin can be produced by fusing to MBP, along with a promoter inversion vector system.

• 한국자기공명학회논문지
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• 제1권1호
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• pp.31-44
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• 1997

The effect of the binding of CRP to the symmetrically synthetic 22, 28, and 30 bp lac promoter was investigated by 1H NMR. The binding of cAMP*CRP to the 22 bp DNA did not bring about any changes in the chemical shift values, but did cause selective line broadening of imino proton resonances of specific base pairs. However, The binding of cAMP*CRP to the 28 and 30 bp DNA brought about large changes on the imino proton resonances that seems to be induced by DNA bending. We studied also the role of cAMP as an activator of DNA/CRP complex formation by gel mobility shift assay. Gel mobility shift assay revealed that the cAMP*CRP complex was not able to bind to the 22 bp DNA fragment, but was able to bind to the 28 bp DNA fragment of lac promoter region.

• Interdisciplinary Bio Central
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• 제3권3호
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• pp.10.1-10.8
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• 2011

Introduction: Hash functions are computer algorithms that protect information and secure transactions. In response to the NIST's "International Call for Hash Function", we developed a biological hash function using the computing capabilities of bacteria. We designed a DNA-based XOR logic gate that allows bacterial colonies arranged in a series on an agar plate to perform hash function calculations. Results and Discussion: In order to provide each colony with adequate time to process inputs and perform XOR logic, we designed and successfully demonstrated a system for time-delayed bacterial growth. Our system is based on the diffusion of ${\ss}$-lactamase, resulting in destruction of ampicillin. Our DNA-based XOR logic gate design is based on the op-position of two promoters. Our results showed that $P_{lux}$ and $P_{OmpC}$ functioned as expected individually, but $P_{lux}$ did not behave as expected in the XOR construct. Our data showed that, contrary to literature reports, the $P_{lux}$ promoter is bidirectional. In the absence of the 3OC6 inducer, the LuxR activator can bind to the $P_{lux}$ promoter and induce backwards transcription. Conclusion and Prospects: Our system of time delayed bacterial growth allows for the successive processing of a bacterial hash function, and is expected to have utility in other synthetic biology applications. While testing our DNA-based XOR logic gate, we uncovered a novel function of $P_{lux}$. In the absence of autoinducer 3OC6, LuxR binds to $P_{lux}$ and activates backwards transcription. This result advances basic research and has important implications for the widespread use of the $P_{lux}$ promoter.

• 한국미생물·생명공학회지
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• 제19권3호
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• pp.228-234
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• 1991

DNA 자동합성기를 이용하여 오릴고뉴클레오타이드들을 합성하였으며 이로부터 인간 인터루킨-2를 코드하는 유전자를 제조하였다. 합성 유전자의 염기서열은 대장균에서 사용빈도가 높은 코돈을 고려하여 결정하였으며, 이때 인터루킨-2의 아미노산 서열은 변화시키지 않았다. 합성된 유전자는 람다 피엘 프로모터를 사용하여 대장균에서 발현시켰다. 인터루킨-2는 대장균에서 불용성의 침전물로 생성되었다. 생성된 인터루킨-2는 인터루킨-2 요구 배양세포주의 성장을 촉진하였다.