• KSBB Journal
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• 제20권2호
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• pp.88-92
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• 2005

인산화 과정은 serine, threonine, tyrosine에서 발생하는 생화학적 반응으로, 본 연구에서는 다양한 응용의 잠재능력이 있는 phosphoserine, phosphothreonine, phosphotyrosine에 대한 엡타머를 개발하였다. 우선 in vitro selection 방법에 의해 combinatorial chemistry로부터 얻어진 RNA library로부터 이들 phosphoamino acids와 친화도를 가지고 있는 엡타머를 찾아낼 수 있었다. 총 10번의 일련 과정을 통해 phosphoserine에 대해서 2.6 nM의 친화도를 가지고 있는 엡타머를 (SeA-06), phosphothreonine에 대해서는 2.7nM의 친화도를 가지고 있는 엡타머 (TrA-18)를 찾아낼 수 있었고, 이들의 RNA 2차 구조를 각각 예측하여 보았다. 그러나 phosphotyrosine의 경우 짧은 길이의 엡타머가 selection됨으로 내부적으로 구조를 가지는 엡타머는 얻을 수 없었다. o-phosphoserine에 대한 항체가 기존에 보고가 되었으나 유사한 구조를 지닌 o-phosphothreonine에도 비슷한 활성을 보여 이들을 구분할 수 있는 리간드를 찾기 힘들었으나, 본 연구에서는 엡타머를 사용한 특이성 조사에서도 서로를 극명하게 구별할 수 있음을 확인할 수 있었다. 이와 같이 발굴된 엡타머를 사용하여 biochip이나 Proteomics 분석 도구의 응용에 큰 기대효과를 제공할 수 있을 것이라 사료된다.

• 한국잡초학회지
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• 제18권1호
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• pp.76-83
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• 1998

피속 잡초 수집종 33종을 대상으로 RAPD marker를 이용하여 피 수집종 간의 유전적변이를 알아보고, 수집종들을 판별할 수 있는 DNA marker를 선발하기 위하여 본 실험을 수행한 결과를 요약하면 다음과 같다. Operon사에서 제작된 74개의 10-mer RAPD primer 가운데에서 명확한 다형성을 보이는 6개 primer를 선발하였다. 이들 primer로 PCR에서 증폭된 밴드는 31개이었으며 이 가운데 다형성을 나타내는 band는 26개(83.9%)로 나타났다. 피 수집종 간의 유연 관계를 분석한 결과 공시된 피 수집종은 크게 3그룹으로 분류할 수 있었다.

• Applied Biological Chemistry
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• 제42권1호
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• pp.34-38
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• 1999

토마토의 genome내에는 적어도 5개 이상의 PAL유전자 좌가 존재한다는 사실을 이 등(1992)이 이미 genomic Southern blot hybridization으로 확인하여 보고하였다. 그러나 본 연구에서 제작한 genomic DNA libraries를 대상으로 검색한 결과 기존에 보고된 PAL유전자 이외에 약 15 kb 와 10 kb에 해당하는 큰 EcoRI 단편을 확보 할 수 있었다. 이들 단편을 BamHI, HindIII등 9종의 제한효소를 사용하여 Southern blot hybridization을 행한 결과 PAL X1의 경우는 모든 효소에 대하여 2개의 단편이 hybridization 되었으며, 특히 BamHI으로 절단하여 얻은 3개의 단편중 두 개는 PAL5 유전자의 exon 2 부위에서 취한 oligomer(18 mer)와 primer extension 반응이 진행되어서 약 200 bp의 PAL유전자와 아주 높은 상동성을 갖는 염기서열이 확인되었다. 따라서 PAL X1유전자는 2 copy의 유전자가 나란히 존재하거나 아니면 염색체 재배열이 진행된 것으로 추정된다. 이러한 결과는 적어도 7개 이상의 PAL유전자 좌가 토마토 염색체내에 존재하는 것으로 사료된다.

• Archives of Pharmacal Research
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• 제29권10호
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• pp.890-897
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• 2006

In inflammatory responses, induction of cytokines and other immune regulator genes in macrophages by pathogen-associated signal such as lipopolysaccharide (LPS) plays a crucial role. In this study, the gene expression profile changes by LPS treatment in the macrophage/monocyte lineage cell line RAW264.7 was investigated. A 60-mer oligonucleotide microarray of which probes target 32381 mouse genes was used. A reverse transcription-in vitro translation labeling protocol and a chemileuminescence detection system were employed. The mRNA expression levels in RAW264.7 cells treated for 6 h with LPS and the control vehicle were compared. 747 genes were up-regulated and 523 genes were down-regulated by more than 2 folds. 320 genes showing more than 4-fold change by LPS treatment were further classified for the biological process, molecular function, and signaling pathway. The biological process categories that showed high number of increased genes include the immunity and defense, the nucleic acid metabolism, the protein metabolism and modification, and the signal transduction process. The chemokine-cytokine signaling, interleukin signaling, Toll receptor signaling, and apoptosis signaling pathways involved high number of genes differentially expressed in response to LPS. These expression profile data provide more comprehensive information on LPS-target genes in RAW264.7 cells, which will be useful in comparing gene expression changes induced by extracts and compounds from anti-inflammatory medicinal herbs.

• BMB Reports
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• 제35권2호
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• pp.206-212
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• 2002

The NS2B-NS3(pro) polyprotein segment from the dengue virus serotype 2 strain 16681 was purified from overexpressing E. coli by metal chelate affinity chromatography and gel filtration. Enzymatic activity of the refolded NS2B-NS3(pro) protease complex was determined in vitro with dansyl-labeled peptide substrates, based upon native dengue virus type 2 cleavage sites. The 12mer substrate peptides and the cleavage products could be separated by reversed-phase HPLC, and were identified by UV and fluorescence detection. All of the peptide substrates (representing the DEN polyprotein junction sequences at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 sites) were cleaved by the recombinant protease NS2B-NS3(pro). No cleavage was observed with an enzymatically inactive S135A mutant of the NS3 protein, or with a modified substrate peptide of the NS3/NS4A polyprotein site that contained a K2093A substitution. Enzymatic activity was dependent on the salt concentration. A 50% decrease of activity was observed in the presence of 0.1M sodium chloride. Our results show that the NS3 protease activity of the refolded NS2B-NS3(pro) protein can be assayed in vitro with high specificity by using cleavage-junction derived peptide substrates.

• Journal of Microbiology and Biotechnology
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• 제17권5호
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• pp.806-811
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• 2007

Bacillus anthracis is a soil pathogen capable of causing anthrax that is closely related to several environmental species, including B. cereus, B. mycoides, and B. thuringiensis. DNA homology studies showed that B. anthracis, B. cereus, B. mycoides, and B. thuringiensis are closely related, with a high sequence homology. To establish a method to specifically detect B. anthracis in situations such as environmental contamination, we initially performed RAPD-PCR with a 10-mer random primer and confirmed the presence of specific PCR bands only in B. anthracis species. One region specific for B. anthracis was cloned and sequenced, and an internal primer set was designed to amplify a 241-bp DNA fragment within the sequenced region. The PCR system involving these specific primer sets has practical applications. Using lyses methods to prepare the samples for PCR, it was possible to quickly amplify the 241-bp DNA segment from samples containing only a few bacteria. Thus, the PCR detection method developed in this study is expected to facilitate the monitoring of environmental B. anthracis contamination.

• 한국균학회지
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• 제29권2호
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• pp.99-103
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• 2001

RAPD 검정법을 이용하여, 우리 나라에 자생하고 있는 야생 표고균주의 지역간 유전변이에 관해 분석하였다. 사용된 야생 표고균주는 계방산 수집 10개, 오대산 수집 11개, 지리산 수집 11개의 야생 표고 총 32균주를 사용하였다. Genomic DNA를 추출하여 10개의 random primer를 사용하여 PCR 증폭시킨 후 전기영동 한 결과 170개의 밴드가 관찰되었으며, 그중 다형성 밴드는 161개가 검출되었다. 이들 전기영동 상에 나타난 밴드의 유무(1,0)로 cluster 분석을 한 결과, 계방산과 오대산 표고균주들이 하나의 그룹으로, 나머지 지리산 표고균주들이 다른 하나의 그룹으로 형성되었다. 또한 AMOVA 분석결과 세 지역의 집단간 유전적인 차이는 12.5%를 나타내었고, 각 균주들 간에는 87.5%임을 알 수 있었다. 이러한 결과는 계방산과 오대산 균주들은 그룹 내 분화가 큰 것이 주요 원인일 것으로 생각되며, 반면, 지리산 균주들은 계방산과 오대산과는 유전적 격리가 존재함을 알 수 있었다.

• Journal of Plant Biotechnology
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• 제5권2호
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• pp.83-86
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• 2003

A reverse transcription polymerase chain reaction (RT-PCR) protocol was developed using two specific 22-mer primers located in coat protein gene of SPFMV. A 411 bp PCR-product was detected in virus infected plants as well as tissue culture raised sweet potato but not in healthy plants. For optimization of RT-PCR protocol, the optimum crude nucleic acid concentration, annealing temperature, primer concentration and numbers of PCR-cycle for maximum sensitivity and specificity were determined. The optimum condition for RT-PCR was as follows: RT-PCR reaction mixture was one-step mixture, containing 50 pmol of primer, 30 units of reverse transcriptase, 5 units of RNasin, and the crude nucleic acid extracts (200 ng). In RT-PCR, cDNA was synthesized at $42^{\circ}C$ for 45 min before a quick incubation on ice after pre-denaturation at $95^{\circ}C$ for 5 min. The PCR reaction was carried out for 40 cycles at $96^{\circ}C$ for 30 see, $63^{\circ}C$ for 30 sec, $72^{\circ}C$ for 1 min, and finally at $72^{\circ}C$ for 10 min. The viral origin of the amplified product was confirmed by sequencing, with the sequence obtained having $95-98\%$ homology with published sequence data for SPFMV. The benefits of this RT-PCR based detection of SPFMV would be simple, rapid and specific.

• Journal of Microbiology and Biotechnology
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• 제29권10호
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• pp.1522-1542
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• 2019

To adapt to environmental changes and to maintain cellular homeostasis, microorganisms adjust the intracellular concentrations of biochemical compounds, including metal ions; these are essential for the catalytic function of many enzymes in cells, but excessive amounts of essential metals and heavy metals cause cellular damage. Metal-responsive transcriptional regulators play pivotal roles in metal uptake, pumping out, sequestration, and oxidation or reduction to a less toxic status via regulating the expression of the detoxification-related genes. The sensory and regulatory functions of the metalloregulators have made them as attractive biological parts for synthetic biology, and the exceptional sensitivity and selectivity of metalloregulators toward metal ions have been used in heavy metal biosensors to cope with prevalent heavy metal contamination. Due to their importance, substantial efforts have been made to characterize heavy metal-responsive transcriptional regulators and to develop heavy metal-sensing biosensors. In this review, we summarize the biochemical data for the two major metalloregulator families, SmtB/ArsR and MerR, to describe their metal-binding sites, specific chelating chemistry, and conformational changes. Based on our understanding of the regulatory mechanisms, previously developed metal biosensors are examined to point out their limitations, such as high background noise and a lack of well-characterized biological parts. We discuss several strategies to improve the functionality of the metal biosensors, such as reducing the background noise and amplifying the output signal. From the perspective of making heavy metal biosensors, we suggest that the characterization of novel metalloregulators and the fabrication of exquisitely designed genetic circuits will be required.

• The Korean Journal of Physiology and Pharmacology
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• 제27권5호
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• pp.481-491
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• 2023

The β subunits of high voltage-gated calcium channels (HGCCs) are essential for optimal channel functions such as channel gating, activation-inactivation kinetics, and trafficking to the membrane. In this study, we report for the first time the potent blood pressure-reducing effects of peptide fragments derived from the β subunits in anesthetized and non-anesthetized rats. Intravenous administration of 16-mer peptide fragments derived from the interacting regions of the β1 [cacb1(344-359)], β2 [cacb2(392-407)], β3 [cacb3(292-307)], and β4 [cacb4(333-348)] subunits with the main α-subunit of HGCC decreased arterial blood pressure in a dose-dependent manner for 5-8 min in anesthetized rats. In contrast, the peptides had no effect on the peak amplitudes of voltage-activated Ca²⁺ current upon their intracellular application into the acutely isolated trigeminal ganglion neurons. Further, a single mutated peptide of cacb1(344-359)-cacb1(344-359)_K357R-showed consistent and potent effects and was crippled by a two-amino acid-truncation at the N-terminal or C-terminal end. By conjugating palmitic acid with the second amino acid (lysine) of cacb1(344-359)_K357R (named K2-palm), we extended the blood pressure reduction to several hours without losing potency. This prolonged effect on the arterial blood pressure was also observed in non-anesthetized rats. On the other hand, the intrathecal administration of acetylated and amidated cacb1(344-359)_K357R peptide did not change acute nociceptive responses induced by the intradermal formalin injection in the plantar surface of rat hindpaw. Overall, these findings will be useful for developing antihypertensives.