• Journal of Microbiology and Biotechnology
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• 제28권9호
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• pp.1467-1472
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• 2018

In this study, DNA-templated copper nanoclusters (DNA-CuNCs) were used to detect exonuclease III (Exo III) activity, which is important for the diagnosis and therapy of several diseases. The results of this study showed that Exo III was affected by the concentrations of magnesium ions and sodium ions, and its optimal conditions for cleavage were $5mM\;Mg^{2+}$ and less than $25mM\;Na^+$. With a blunt-end DNA, more than 98% of DNA was digested by Exo III. As expected, with two or four cytosines in the terminal position of a 4-base overhanging DNA such as 5'-GGCC-3' and 5'-CCCC-3', there was little cleavage by Exo III compared with a blunt-end DNA.

• Journal of Microbiology and Biotechnology
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• 제33권12호
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• pp.1692-1697
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• 2023

Staphylococcus aureus integrated with mecA gene, which codes for penicillin-binding protein 2a, is resistant to all penicillins and other beta-lactam antibiotics, resulting in poor treatment expectations in skin and soft tissue infections. The development of a simple, sensitive and portable biosensor for mecA gene analysis in S. aureus is urgently needed. Herein, we propose a dual-toehold-probe (sensing probe)-mediated exonuclease-III (Exo-III)-assisted signal recycling for portable detection of the mecA gene in S. aureus. When the target mecA gene is present, it hybridizes with the sensing probe, initiating Exo III-assisted dual signal recycles, which in turn release numerous "3" sequences. The released "3" sequences initiate catalytic hairpin amplification, resulting in the fixation of a sucrase-labeled H2 probe on the surface of magnetic beads (MBs). After magnet-based enrichment of an MB-H1-H2-sucrase complex and removal of a liquid supernatant containing free sucrase, the complex is then used to catalyze sucrose to glucose, which can be quantitatively detected by a personal glucose meter. With a limit of detection of 4.36 fM for mecA gene, the developed strategy exhibits high sensitivity. In addition, good selectivity and anti-interference capability were also attained with this method, making it promising for antibiotic tolerance analysis at the point-of-care.

• Journal of Microbiology and Biotechnology
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• 제8권5호
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• pp.471-477
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• 1998

Taq DNA polymerase from Thermus aquaticus is very useful in the polymerase chain reaction. Taq DNA polymerase is classified in the pol I family, represented by E. coli DNA polymerase I. The three-dimensional structural alignment of 3'-5'exonuclease domains from the pol I family DNA polymerases explains why Taq DNA polymerase does not carry out proofreading in polymerase chain reactions. Three sequence motifs, Exo I, II, and III, must exist to carry out 3'-5'exonuclease activity for proof- reading by a 3'-5'exonuclease reaction, but these are abolished in Taq DNA polymerase. The key catalytic module in 3'-5'exonuclease is two metal ions chelated by four active-site carboxylic amino acids. Taq DNA polymerase was mutagenized to construct the catalytic module in the active site. The circular dichroism technique supported the formation of the catalytic module, and the radioactive assay showed that the 3'-5'exonuclease activity doubled in the mutant Taq DNA polymerase.

• 대한화학회지
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• 제65권5호
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• pp.397-400
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• 2021

• 미생물학회지
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• 제31권2호
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• pp.141-147
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• 1993

백색 부후균인 plaurotus ostreatus 의 4가지의 균주로부터 각각 10.2 kb 와 7.2 kb (NFFA 2), 두 종류의 10.2 kb (NFFA 4001) 11.2 kb (NFFA 4501), 10.2kb 와 11.2 kb(KFCC 11635) 크기의 미토콘드리아 플라스미드들을 분리해 내었다. NFFA 2 의 변종인 NFFA 2m1 과 NFFA 2m2 에서는 이들 플라스미드가 관찰되지 않았다. 분별 원심분리에 의해 얻은 미토콘드리아에서 핵산을 추출하여 agarose gel 에서 전기영동시키면 플라스미드가 관찰되지 않았으나 proteinase K 를 처리하고 핵산을 추출하여 전기영동한 결과 이들 플라스미드가 관찰되었는에, 이는 플라스미드상에 단백질인 결합되어 있음을 시사한다. Proteinase K 를 처리한 플라스미드 DNA 와 exonuclease 를 반응시킨 결과, 이들 플라스미드들은 5'말단에 단백질이 결합된 성형 이중가닥 DNA의 구조를 가진 것을 확인하였다. 각 플라스미드들의 상호관계를 조사하기 위하여 Southern hybridization 을 수행한 결과 최소 3가지 종류의 플라스미드들로 분류할 수 있었다. 이중 한 그룹 (group I) 은 모든 P. ostreatus 균주들에서 공통적으로 발견되었다. Pleurotus 속의 5가지 다른 종(P. cornucopiae, P. florida, P. pulmonarius, P. sajor-cuja, P. spodoleucus) 의 균주들로부터 미토콘드리아 플라스미드들을 분리하였다. 이들은 한균주당 1-4 개 까지의 플라스미드를 가지며, 플라스미드의 크기는 7.2 kb-14kb 범위에 있었다. P. ostreatus 의 NFFA 2 의 10.2 kb(group I) 플라스미드와 hybridization 을 수행한 결과 P. cornucopiae ASI 2011을 제외한 다른 모든 균주들이 유사한 염기서열의 플라스미드를 갖고 있음을 알았다.

• BMB Reports
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• 제30권3호
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• pp.188-193
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• 1997

Membrane lipid peroxidation processes yield products that may react with DNA to cause mutations. Lipid hydroperoxides from linoleic acid in the presence of transition metal ions caused strand breaks in plasmid DNA. DNA damage induced by reactive aldehydes known to be produced by decomposition of lipid hydroperoxides, such as 4-hydroxynonenal or rnalondialdehyde, was repaired by endonucleases and exonuclease III which resulted in the increase of single strand breaks in DNA. Lipid hydroperoxides as well as malondialdehyde and 4-hydroxynonenal also caused mutations in the pUC18 lacZ' gene when measured as a loss of ${\alpha}-cornplementation$. In conclusion. the lipid peroxidation could be an important intermediary event in DNA damage and mutation by oxidative stress.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1994년도 춘계학술대회 and 제3회 신약개발 연구발표회
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• pp.192-192
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• 1994

본 논문에서는 cytochrome P450 LA1 유전자의 5'-upsteam 조절부위의 클로닝을 실시하였다. pUC19 vector에 연결시킨 3.4 Kb 크기의 Pstl DNA조각을 Sst1, Nco1 제한 효소로 자른 뒤, Exonuclease III 를 처리하여 약 200bp 씩의 차이를 갖는 여러 크기의 plasmid들을 얻었다. 이 plasmid 의 핵산서열을 알아보기 위해 dideoxy nucletide를 이용한 sequencing방법으로 그 핵산서열의 결정 실험을 시도하였다. 또한, 다환상 방향족 탄화수소 화합물에 반응성을 갖는 C57BL/6N 생쥐와 반응성을 갖지않는 DBA/2N 생쥐에 있어 phase II 대사 효소인UDP-glucuronosyltransferase 효소활성에 대한 3-methylcholanthrene의 영향을 알아보기 위해 C57BL/6N 생쥐와 DBA/2N 생쥐에 각각 다른 농도의 3-methylcholanthrene을 처리하거나 각기 다른 시간에 3-methylcholanthrene를 처리하였다. 그 결과 UDP-glucuronosyl-transferase의 mRNA가 3-methylcholanthrene양의 증가에 따라, 처치시간이 길어짐에 따라 증가되어지며 그 mRNA위 크기는 약 2.2Kb 정도임을 알았다. 이로부터 UDP-ghucuronosyltransferase 또한 cytochrome P45O와 함께 다환상 방향족 탄화수소 화합물 조절인자를 통한 조절을 받을 것이며 phase I phase II 약물 대사 효소가 조절상 밀접한 관련을 가짐을 예측할 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제9권1호
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• pp.91-97
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• 1999

In this work, a 1.9-kb region of enterococcal plasmid p703/5 was isolated and the nucleotide sequence analysis of the region was performed. One major open reading frame (ORF) was identified encoding a polypeptide of 28 kDa. Database comparisons suggested that the protein showed some homology with other bacterial RepA proteins. Upstream of the ORF, a potential dnaA box, AT-rich region and 22-bp tandemly repeated sequences (DNA iterons), a feature typical for many replication ori sites, were recognized. Deletion analysis using Exonuclease III and several restriction enzymes indicated that the three elements and the gene product from the ORF were essential for replication and that the minimum unit of DNA required for replication resided on the 1.2-kb AvaII subfragment. Thus, this gene product was referred to as RepA.

• Archives of Pharmacal Research
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• 제19권6호
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• pp.450-455
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• 1996

The genomic DNA was prepared from trout liver which was treated with 3-methycholanthrene, and cloned into lambda EMBL3 at BamHl site. The genomic library was constructed via infections of these recombinant phages into E. coli K802, and screened by the most $5^I$-portion of trout CYP1A1 cDNA. After the screening of $10^9$ clones of the amplified library, 12 positive clones were isolated, and subjected to further screenings. The results of southern blot hybridization of genomic DNA prepared from the positive clone showed the presence of a single gene of CYP1A1, and 3.5 Kb PstI fragment that hybridizes with the most $5^I$-region DNA of CYP1A1 cDNA. The restriction map of PstI fragment was determined by the restriction digestion with various enzymes. The nucleotide sequence of the upstream genomic DNA of CYPIAI was determined by DNA sequencing of exonuclease III unidirectionally deleted PstI fragment DNA using $[^{35}/S]$dATP. This paper presented the upstream genomic DNA of CYP1A1 contained a part of coding region which was about 351 base pairs (from ATG to PstI site at 3563).

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.71-71
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• 2002

Promoters for milk proteins have been used far producing transgenic animals due to their temporal and spatial expression patterns. ${\beta}$-casein, a calcium-sensitive casein, is a major milk protein that corresponds ca. 30 per cent of total milk protein. Expression of ${\beta}$-casein is controlled by lactogenic hormones such as prolactin (PRL), composite response elements (CoREs) and transcription factors. CoREs are clusters of transcription factor binding sites containing both positive and negative regulatory elements. ${\beta}$-casein gene promoter contains various regions (CoREs) for gene transcription. We analyzed the promoter region by mutagenesis using exonuclease III and linker-scanning. Transcription control elements usually are positioned in 5'-flanking region of the gene. However, in some cases, these elements are located in other regions such as intron 1. The nucleotide sequences of ${\beta}$-casein promote. region has been reported (E12614). However, the properties of the promoter is not yet clear. In this study, we plan to investigate the properties of cis-regulating elements of porcine ${\beta}$-casein by mutation analysis and expression analysis using dual-luciferase repoter assay system.