• BMB Reports
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• 제30권1호
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• pp.41-45
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• 1997

The present study was performed to analyze the molecular mechanism which dictates the transcription regulation of the $O^6$-methylguanine-DNA methyltransferase (MGMT) gene in Saccharomyces cerevisiae. Previously we identified one possible upstream repressing sequence (URS) in MGMT promoter by promoter deletion and competition analysis. In this paper we report another regulatory element (UAS: upstream activating sequence. -213 to -136) which affects the transcription activity of MGMT promoter. Gel mobility shift assay and Southwestern blot analysis using UAS probe showed several specific proteins which were able to bind to this sequence.

• BMB Reports
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• 제54권2호
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• pp.98-105
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• 2021

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a family of DNA sequences originally discovered as a type of acquired immunity in prokaryotes such as bacteria and archaea. In many CRISPR systems, the functional ribonucleoproteins (RNPs) are composed of CRISPR protein and guide RNAs. They selectively bind and cleave specific target DNAs or RNAs, based on sequences complementary to the guide RNA. The specific targeted cleavage of the nucleic acids by CRISPR has been broadly utilized in genome editing methods. In the process of genome editing of eukaryotic cells, CRISPR-mediated DNA double-strand breaks (DSB) at specific genomic loci activate the endogenous DNA repair systems and induce mutations at the target sites with high efficiencies. Two of the major endogenous DNA repair machineries are non-homologous end joining (NHEJ) and homology-directed repair (HDR). In case of DSB, the two repair pathways operate in competition, resulting in several possible outcomes including deletions, insertions, and substitutions. Due to the inherent stochasticity of DSB-based genome editing methods, it was difficult to achieve defined single-base changes without unanticipated random mutation patterns. In order to overcome the heterogeneity in DSB-mediated genome editing, novel methods have been developed to incorporate precise single-base level changes without inducing DSB. The approaches utilized catalytically compromised CRISPR in conjunction with base-modifying enzymes and DNA polymerases, to accomplish highly efficient and precise genome editing of single and multiple bases. In this review, we introduce some of the advances in single-base level CRISPR genome editing methods and their applications.

• Journal of Microbiology
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• 제43권3호
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• pp.308-312
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• 2005

The global RNA transcription profiles of Bacillus lentimorbus WJ5 under an in vitro co-culture with Colletotrichum gloeosporioides were analyzed in order to study the antagonistic bacteria-fungi interactions. Using a filter membrane system, B. lentimorhus WJ5 was exposed to the spores of C. gloeosporioides at the late exponential stage. The transcription profiles of the B. lentimorhus WJ5, both with and without a challenge from C. gloeosporioides, were analyzed using custom DNA chips containing 2,000 genome fragments. A total of 337 genes were expressed, with 87 and 47 up- and down-regulated, respectively. Of these, 12 genes, which were involved in central carbon metabolisms, and 7 from minor catabolism were relatively highly up-regulated (> 10 fold) and down-regulated (< 0.2 fold), respectively. Nine genes, which were thought to be related to the antifungal activity, were also up-regulated, but their levels were not so high (2.0 - 9.7 folds). From the results, during the early stage of the co-culture of B. lentimorbus WJ5 and C. gloeosporioides, nutrient competition seemed to occur; therefore, the genes from central carbon metabolisms could be up-regulated, while those from minor catabolism could be down-regulated.

• BMB Reports
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• 제32권1호
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• pp.45-50
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• 1999

We previously found three transcription factor-binding motifs in the rat p53 promoter. They are two recognition motifs of NF1-like protein (NF1-like element 1: -296 ~ -312, NF1-like element 2: -195 ~ -219) and a bHLH protein binding element (-142 ~ -146). In this study, we investigated the DNA-protein complex formation of the three elements with nuclear extracts from both normal and regenerating liver to find the element involved in the induced transcription of p53. The level of each DNA-protein complex on NF1-like and bHLH motifs was not changed. Instead, a new element located at -264 ~ -284 was detected in the DNase I footprinting assay with regenerating nuclear extract. This element has partial homology to the AP1 consensus motif. However, the competition studies with diverse oligonucleotides suggest that the binding protein is not AP1. An in vitro transcription assay shows that this element is important for the transcriptional activation of the rat p53 promoter. Therefore, for the induced transcription of the rat p53 promoter, the-264 ~ -284 region is required in addition to two NF1-like and one bHLH motif.

• 대한의생명과학회지
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• 제16권3호
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• pp.201-206
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• 2010

In order to develop procedures for the rapid isolation of recombinant sugar transporter in functional form from away from the endogenous insect cell transporter, gene fusion techniques were exploited. Briefly, BamH1-digested human HepG2 type glucose transport protein cDNA was first cloned into a transfer vector pBlueBacHis, containing a tract of six histidine residues. Recombinant baculoviruses including the human cDNA were then generated by allelic exchange following transfection of insect cells with wild-type BaculoGold virus DNA and the recombinant transfer vector. Plaque assay was then performed to obtain and purify recombinant viruses expressing the human transport protein. All the cell samples that had been infected with viruses from the several blue plaques exhibited a positive reaction in the immnuassay, demonstrating expression of the glucose transport protein. In contrast, no color development in the immunoassay was observed for cells infected with the wild-type virus or no virus. Immunoblot analysis showed that a major immunoreactive band of apparent Mr 43,000~44,000 was evident in the lysate from cells infected with the recombinant baculovirus. Following expression of the recombinant fusion protein with the metal-binding domain and enterokinase cleavage site, the fusion protein was recovered by competition with imidizole using immobilized metal charged resin. The leader peptide was then removed from the fusion protein by cleavage with porcine enterokinase. Final separation of the recombinant protein of the interest was achieved by passage over $Ni^{2+}$-charged resin under binding conditions. The expressed transport protein bound cytochalasin B and demonstrated a functional similarity to its human counterpart.

• Parasites, Hosts and Diseases
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• 제53권2호
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• pp.147-154
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• 2015

Various constituents in clinical specimens, particularly feces, can inhibit the PCR assay and lead to false-negative results. To ensure that negative results of a diagnostic PCR assay are true, it should be properly monitored by an inhibition control. In this study, a cloning vector harboring a modified target DNA sequence (${\approx}375bp$) was constructed to be used as a competitive internal amplification control (IAC) for a conventional PCR assay that detects ${\approx}550bp$ of the Cryptosporidium oocyst wall protein (COWP) gene sequence in human feces. Modification of the native PCR target was carried out using a new approach comprising inverse PCR and restriction digestion techniques. IAC was included in the assay, with the estimated optimum concentration of 1 fg per reaction, as duplex PCR. When applied on fecal samples spiked with variable oocysts counts, ${\approx}2$ oocysts were theoretically enough for detection. When applied on 25 Cryptosporidium-positive fecal samples of various infection intensities, both targets were clearly detected with minimal competition noticed in 2-3 samples. Importantly, both the analytical and the diagnostic sensitivities of the PCR assay were not altered with integration of IAC into the reactions. When tried on 180 randomly collected fecal samples, 159 were Cryptosporidium-negatives. Although the native target DNA was absent, the IAC amplicon was obviously detected on gel of all the Cryptosporidium-negative samples. These results imply that running of the diagnostic PCR, inspired with the previously developed DNA extraction protocol and the constructed IAC, represents a useful tool for Cryptosporidium detection in human feces.

• Applied Biological Chemistry
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• 제48권3호
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• pp.296-300
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• 2005

Among the root colonizing and plant growth promoting bacteria isolated from the bacterial wilt suppressive soil, five strains were detected to produce siderophores by CAS agar assay. The most effective isolate, TS3-7 strain induced significant suppression of bacterial wilt disease in tomato and pepper plants. Seed treatment followed by soil drench application with this strain resulted in over 80% reduction of bacterial wilt disease compared with the control. Significant disease suppression by TS3-7 strain was related to the production of siderophore. Besides iron competition, induction of resistance of the host plant with siderophore was suggested to be another mode of action that suppress bacterial wilt, based on the lack of direct antibiosis against pathogen in vitro. According to Bergey's Manual of Systemic Bacteriology and 16S rDNA sequence data, TS3-7 stain was identified as Pseudomonas sp. TS3-7.

• 생명과학회지
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• 제28권12호
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• pp.1432-1437
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• 2018

클라라 세포에 의해 생산되는 클라라 세포 분비 단백질(CCSP)은 폐를 염증으로부터 보호하는데 중요한 역할을 한다. 이 연구는 CCSP 유전자 발현에 관여하는 프로모터 부위에서 repressor에 결합할 수 있는 cis-element를 밝히는데 있다. DNaseI footprinting법을 사용하여 mCCSP 프로모터의 -812에서 -768 bp (45 bp) 사이에서 3 개의 보호된 motif를 찾았고, 그 중 하나인 D3 모티프(GCCTGGGAA)는 다른 3 가지 동물들과 염기서열이 100% 일치하였다. 45 bp를 사용한 EMSA 분석에서 D3 모티프(GGCCTGGGAA)는 45 bp에 높은 경쟁을 보였으나, 변이된 D3 모티프가 ($G{\underline{AA}}TG{\underline{TT}}AA$)를 사용되었을 때, 경쟁은 상당히 감소되었다. 이는 mCCSP 프로모터의 45 bp의 D3 모티프가 단백질과 DNA 상호 작용을 위한 중요한 element임을 시사한다. -756-Luc과 -812-Luc을 이용한 transient transfection 분석 결과, -756-Luc은 -812-Luc보다 CCSP의 발현이 현저하게 감소되었다. 이는 mCCSP 프로모터의 45 bp부위가 repressor의 결합 부위로서 기능을 할 수 있음을 의미한다. -812-Luc에 KLF4를 co-transfection 한 결과, KLF4는 CCSP 발현을 현저하게 저해(repression)함을 밝혔다. 그러나 -768-Luc이 사용되었을 때 KLF4에 의한 repression은 관찰되지 않았다. 이것은 KLF4가 CCSP 유전자의45 bp에 결합할 수 있고, 전사 억제자 역할을 하여 mCCSP 발현을 억제 할 수 있음을 명확히 보여 준다. 또한 이는 45 bp 중, D3 모티프가 KLF4의 결합에 강하게 관여 함을 시사한다. 이 반응에 KLF4에 대한 항체가 첨가되었을 때는 super-shifted 밴드가 관찰되었으나, SP1에 대한 항체가 사용되었을 때는 관찰되지 않았다. 이는 KLF4가 CCSP 프로모터의 45 bp 영역에 결합하여 repressor기능 할 수 있고, D3 모티프가 KLF4의 특이적 결합에 관여 할 수 있음을 시사한다.

• Biomolecules & Therapeutics
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• 제4권1호
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• pp.103-109
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• 1996

To investigate the effect of the third intracellular loop (i3 loop) peptide of human $\beta$$_2$-adrenergic receptor on receptor agonist binding, we expressed third intracellular loop region of human $\beta$$_2$-adrenergic receptor as glutathione S-transferase fusion protein in E. coli. DNA fragment of the receptor gene which encodes amino acid 221-274 of human $\beta$$_2$-adrenergic receptor was amplified by polymerase chain reaction and subcloned into the bacterial fusion protein expression vector pGEX-CS and expressed as a form of glutathione-S-transferase (GST) fusion protein in E. coli DH5$\alpha$. The receptor fusion protein was identified by SDS-PAGE and Western blot using monoclonal anti-GST antibody. The fusion protein expressed in this study was purified to an apparent homogeneity by glutathione Sepharose CL-4B affinity chromatography. The purified i3 loop fusion proteins at a concentration of 10 $\mu\textrm{g}$/ι caused right shift of the isoproterenol competition curve of [$^3$H]Dihydroalprenolol binding to hamster lung $\beta$$_2$-adrenergic receptor indicating lowered affinity of isoproterenol to $\beta$$_2$-adrenergic receptor possibly due to the uncoupling of receptor and G protein in the presence of the fusion protein. The uncoupling of receptor and G protein suggests that i3 loop region plays a critical role on $\beta$$_2$-adrenergic receptor G protein coupling.

• Journal of Ecology and Environment
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• 제47권4호
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• pp.168-176
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• 2023

Background: Many insectivorous bats have flexible diets, and the difference in prey item consumption among species is one of the key mechanisms that allows for the avoidance of interspecies competition and promotes coexistence within a microhabitat. In Korea, of the 24 bat species that are known to be distributed, eight insectivorous bats use forest areas as both roosting and foraging sites. Here, we aimed to understand the resource partitioning and coexistence strategies between two bat species, Myotis ikonnikovi and Plecotus ognevi, cohabiting the Mt. Jumbong forests, by comparing the differences in dietary consumption based on habitat utilization. Results: Upon examining their dietary composition using the DNA meta-barcoding approach, we identified 403 prey items (amplicon sequence variants). A greater prey diversity including Lepidoptera, Diptera, Coleoptera, and Ephemeroptera, was detected from M. ikonnikovi, whereas most prey items identified from P. ognevi belonged to Lepidoptera. The diversity index of prey items was higher for M. ikonnikovi (H': 5.67, D: 0.995) than that for P. ognevi (H': 4.31, D: 0.985). Pianka's index value was 0.207, indicating little overlap in the dietary composition of these bat species. Our results suggest that M. ikonnikovi has a wider diet composition than P. ognevi. Conclusions: Based on the dietary analysis results, our results suggests the possibility of differences in foraging site preferences or microhabitat utilization between two bat species cohabiting the Mt. Jumbong. In addition, these differences may represent one of the important mechanism in reducing interspecific competition and enabling coexistence between the two bat species. We expected that our results will be valuable for understanding resource partitioning and the coexistence of bats inhabiting the Korean forests.