• Restorative Dentistry and Endodontics
• /
• 제20권2호
• /
• pp.645-654
• /
• 1995

Porphyromonas endodontalis is a black-pigmented anaerobic Gram negative rod which is associated with endodontal infections. It has been isolated from infected dental root canals and submucous abscesses of endodontal origin. DNA probe is an available alternative, offering the direct detection of a specific microorganism. Nucleic-acid probes can be off different types: whole different: whole-genomic, cloned or oligonucleotide probes. Wholegenomic probes are the most sensitive because the entire genome is used for possible hybridization sites. However, as genetically similar species of bacteria are likely to be present in specimences, cross-reactions need to be considered. Cloned probes are isolated sequences of DNA that do not show cross-reactivity and are produced in quantity by cloning in a plasmid vector. Cloned probes can approach the sensitivity found with whole-genomic probes while avoiding known cross-reacting species. Porphyromonas endodontalis ATCC 35406 (serotype $O_1K_1$) was selected in this experiment to develop specific cloned DNA probes. EcoR I-digested genomic DNA fragments of P. endodontalis ATCC 35406 were cloned into pUC18 plasmid vector. From the E. coli transformed with the recombinant plasmid 4 clones were selected to be tested as specific DNA probes. Restriction-digested whole-genomic DNAs prepared from P. gingivalis 38(serotype a), W50(serotype b), A7A1-28(serotype C), P. intermedia 9336(serotype b), G8-9K-3(serotype C), P. endodontalis ATCC 35406(serotype $O_1K_1$), A. a Y4(serotype b), 75(serotype a), 67(serotype c), were each seperated on agarose gel electrophoresis, blotted on nylon membranes, and were hybridized with digoxigenin-dUTP labeled probe. The results were as follows: 1. Three clones of 1.6kb(probe e), 1.6kb(probe f), and 0.9kb(probe h) in size, were obtained. These clones were identified to be a part of the genomic DNA of P. endodontalis ATCC 35406 judging from their specific hybridization to the genomic DNA fragments of their own size on Southern blot. 2. The clones of 4.9kb(probe i) was identified to be a part of the genomic DNA of P. endodontalis ATCC 35406. but not to specific for itself. It was hybridized to P. gingivalis A7A1-28, P. intermedia G89K-3.

• 한국식물병리학회지
• /
• 제14권2호
• /
• pp.177-183
• /
• 1998

A DNA fragment which is involved in tolassin production was cloned to obtain a molecular marker of Pseudomonas tolaasii, a casual agent of bacterial brown blotch disease of oyster mushroom (Pleurotus ostreatus). Tolaasin is a lipodepsipeptide toxin and known as a primary disease determinant of the P. tolaasii. It is responsible for formation of white line in agar when P. tolaasii were cultured against white line reacting organisms (WLROs). White line negative mutants (WL-) were generated by conjugation between rifampicin resistant strain of P. tolaasii and E. coli carrying suicidal plasmid pSUP2021 : : Tn5. The ability of tolaasin production of the WL- mutants was examined by hemolysis test, pathogenicity test, and high pressure liquid chromatography (HPLC) analysis of culture filtrate. All of the WL- mutants were lost the ability of tolaasin production (Tol-). Genomic library of the Tol- mutant was constructed in pLAFR3 and the cosmid clone containing Tn5 was selected. DNA fragment fro franking region of Tn5 was cloned from the plasmid and used as a probe in Southern blot. DNA-DNA hybridization with the probe to total DNA from group of bacteria ecologically similar to P. tolaasii including WLORs, fluorescent Pseudomonads isolated from oyster mushroom, P. agarici, P. gingeri, and some of other species of Psedomonas showed that some of the tested bacteria do not have any hybridized band and others have bands sowing RFLP. The cloned DNA fragment or its nucleotide sequence will be useful in detection and identification of the P. tolaasii.

• 대한화장품학회지
• /
• 제21권1호
• /
• pp.53-66
• /
• 1995

우유 단백질인 ${\gamma}$- 카제인 유전자는 여러 호르몬들에 의해서 임신기간과 비유기기간 동안에 동물의 유선조직에서 발현되는 카제인 유전자 집단의 하나이다. 우유 단백질 유전자의 유도를 조절하는 호르몬에 관한 메커니즘 설명으로 생쥐 ${\gamma}$- 카제인 유전자가 분석되었고 특성을 밝혔다. ${\gamma}$- 카제인 유전자는 박테리오파지 EMBL 3벡터에 삽입된 제놈 도서관으로부터 ${\gamma}$- 카제인 cDNA를 probe로 사용하여 스크린하여 하나의 클론을 얻었다. ${\gamma}$- 카제인 cDNA를 probe는 부분적으로 염기배열을 밝혔으며 ATC 개시 암호와 5'-noncoding 부위를 포함하고 있다. 클론된 제놈 DNA는 제한효소 Sal I에 의해서 ENBL 3벡터로부터 분리 되었다. 3개의 DNA밴드들을 관찰할 수 있었다. 각각의 크기는 28Kb, 14Kb 그리고 9Kb 이다. 따라서 삽입된 DNA의 크기는 대략적으로 23Kb 이다. Southern blot 분석 결과, 클론된 제놈 DNA는 cDNA 5' 발단 부위를 합성한 oilgonucleotides(40 mer)와는 결합되지 않음을 보여주고, 그러나 ${\gamma}$- 카제인 cDNA와는 결합되는 것을 보여 준다. Pormoter 부위를 포함하는 ${\gamma}$- 카제인 제놈 DNA는 cDNA 5' 말단 부위의 합성된 probe에 의해서 생쥐 제놈 도서관으로 부터 스크린하여 현재 29개의 클론을 얻었다.

• 한국식물병리학회지
• /
• 제14권2호
• /
• pp.164-170
• /
• 1998

This study was carried out to develop DNA probe for specific detection of Erwinia carotovora subsp. carotovora. Universal rice primer (URP, 20 mer) developed from repetitive sequence of rice was applied for producing PCR DNA fingerprints of Erwinis spp. In E. carotovora subsp. carotovora strains, primer URP2F amplyfied polymorphic bands which are distinguisable from other Erwinia spp. A PCR band of 0.6 kb selected from PCr polymorphic bands of E. carotovora subsp. carotovora strains was cloned and evaluated as a diagnostic DNA probe. Among 28 bacterial strains including 22 Erwinia spp, the probe (pECC2F) only hybridized to total DNAs from e. carotovora subsp. carotovora strains and E. carotovora subsp. wasabiae, but sizes of hybridized bands were different between these subspecies, 10.0 kb and 3.5 kb respectively. In dot blot assays using probe pECC2F, as few as 103 colony forming units (CFU) of E. carotovora subsp. carotovora could be detected in a suspension containing about 1$\times$103 CFU of soil bacteria.

• Journal of Microbiology and Biotechnology
• /
• 제5권4호
• /
• pp.194-199
• /
• 1995

A portion (7.4 kb) of ribosomal DNA tandem repeat unit from a genome of the mushroom T. matsutake has been cloned. A 1.75 kb EcoRI fragment was cloned first using S. cerevisiae 255 rRNA gene as a probe, and this was then used for further cloning. A chromosomal walking experiment was carried out and the upstream region of the 1.75 kb fragment was cloned using SmaI/BamHI enzyme, the size was estimated to be 5.2 kb in length. Part of the downstream region of the 1.75 kb fragment was also cloned using XbaI/BamHI enzymes. Restriction enzyme maps of three cloned DNA fragments were constructed. Northern hybridization, using total RNA of T. matsutake, and the restriction fragments of three cloned DNAs as probes, revealed that all four ribosomal RNA genes (large subunit[LSU], small subunit [SSU], 5.85 and 5S rRNA genes) are present in the cloned region. The gene organization of the rDNA are regarded as an intergenic spacer [IGS]2 (partial) - SSU rRNA - internal transcribed spacer [ITS]1 - 5.8S rRNA - ITS2 - LSU rRNA - IGS1 -5S rRNA - IG52 (partial).

• 미생물학회지
• /
• 제42권2호
• /
• pp.89-94
• /
• 2006

본 연구는 치주질환 병인론 연구에 빈번히 사용되는 Prevotella intermedia ATCC 49046 균주를 특이적으로 검출 및 동정할 수 있는 PCR primer를 개발하기 위하여 시행하였다. P. intermedia ATCC 49046 유전체 DNA를 추출하고, Hind III 제한효소를 이용하여 무작위 클로닝법으로 유전체 DNA 절편을 얻었다. Southern blot 분석법을 이용하여 DNA 절편의 특이성을 조사하였고, chain termination 법을 이용하여 핵산염기서열을 결정하였다. 이를 바탕으로 PCR primer를 설계하고, P. intermedia ATCC 49046에 대한 균주 특이성 및 검출 한계(민감도)를 조사하였다. Southern blot 분석 결과 Pig6 DNA probe는 서양인에서 분리 동정된 P. intermedia 균주와만 hybridization하였고, 한국인에서 분리 동정된 P. intermedia균주들과는 반응이 없었다. Pig6 DNA probe는 813 bp의 핵산염기로 구성되어 있었으며, 이를 바탕으로 설계된 Pig6-F3와 Pig6-R3 primer 쌍에 의해서는 서양 균주에 특이적인 PCR산물이 증폭되었다. Pig6-60F와 Pig6-770R primer 쌍에 의해서는 P. intermedia ATCC 49046 유전체 DNA에서만 특이적인 PCR 산물이 증폭되었다. 두 가지 primer 쌍들 각각에 대한 P. intermedia 유전체 DNA량의 검출 한계를 알아보기 위한 민감도실험 결과 두가지 primer 쌍들 모두 4 pg (약2000마리)까지 검출 가능하였다. 이상의 연구결과를 종합하면, Pig6-60F와 Pig6-770R primer쌍은 P. intermedia ATCC 49046을 균주 특이적으로 동정할 수 있어, 이 균주의 보존적 측면에서 유용하게 이용될 수 있을 것으로 사료된다.

• Parasites, Hosts and Diseases
• /
• 제44권3호
• /
• pp.233-237
• /
• 2006

We postulated that apolysis was processed in accordance with apoptotic changes occurring in a cestode, Spirometra erinacei (Pseudophyllidea). We cloned the novel putative apoptosis-associated gene from S. erinacei via screening of a S. erinacei cDNA library with a ced-3 gene (activator of apoptosis) probe from Caenorhabditis elegans. We identified a 261-bp cDNA sequence, which encodes for an 86-amino acid protein. The cloned gene expression was observed in the neck and gravid proglottids via Northern blotting, using cloned cDNA inserts as probes, but the clone was not expressed in any of other tissues. We suggest that this gene may be involved in the apolysis of S. erinacei during normal tissue development and differentiation in cestode parasites.

• Journal of Periodontal and Implant Science
• /
• 제26권2호
• /
• pp.365-375
• /
• 1996

5 serotypes(a, b, c, d, e) of Actinobacillus actinomycetemcomitans showed distinct hybridization patterns(DNA fingerprinting patterns) when the bacterial DNA were hybridized with randomly cloned 4.7-Kb sized DNA probe. The sizes of hybridized bands in each serotypes were different among serotypes and represented unique patterns of hybridization with the probe used. The serotype a showed two bands of fingerprinting patterns: 23.1 kb and 2.5 kb respectively. Serotype b and c showed single band: 6.6 kb and 9.5 kb, respectively. Serotype d and e showed two bands of hybridization: 23.1 kb and 2.8 kb, and 23.7 kb and 2.1 kb, respectively. The results indicate that this standard fingerpriting patterns of DNA hybridization with 4.7 kb probe can be further used for genotyping clinical isolates of Actinobacillus 8ctinomycetemcomitansand its relevance with periodontal disease activity.

• Journal of Microbiology and Biotechnology
• /
• 제1권1호
• /
• pp.37-44
• /
• 1991

A shuttle promoter-probe vector, pEB203, was derived from pBR322, pPL703 and pUB110. Using the vector, a useful DNA fragment, 319 bp EcoRI fragment, having strong promoter activity has been cloned from Bacillus subtills chromosomal DNA. Selection was based on chloramphenicol resistance which is dependent upon the introduction of DNA fragments allowing expression of a chloramphenicol acetyl transferase gene. The nucleotide sequence of the 319 bp fragment has been determined and the putative -35 and -10 region, ribosome binding site, and ATG initiation codon were observed. This promoter was named EB promoter and the resultant plasmid which can be used as an expression vector was named pEBP313. The crystal protein gene from B. thuringiensis subsp. kurstaki HD-73 was cloned downstream from the EB promoter without its own promoter. When the resultant plasmid, pBT313, was introduced into Escherichia coli and B. subtilis, efficient synthesis of crystal protein was observed in both cells, and the cp gene expression in B. subtilis begins early in the vegetative phase. The cell extracts from both clones were toxic to Hyphantria cunea larvae.

• 한국수정란이식학회지
• /
• 제10권1호
• /
• pp.91-100
• /
• 1995

3.3kb 웅성특이 DNA(pEM39 plasmid DNA)가 성 특이 DNA 검색자로 활용되어질 수 있는가를 확인하기 위하여 구조적인 분석을 Southern blotting, DNA sequencing과 computer program 분석을 통하여 실시하였다. 전체 3.3kb에서 유래된 약 1kb 단위의 단편을 이용하여 표지된 짧은 DNA probe들은 Southern blot 분석에서 웅성특이성을 나타내었다. McGraw와 Jeon의 sequence에 대한 유사성 비교 자료로부터 여러 부분의 conserved region을 찾아내고 이것을 기초로 하여 5개의 primer set들을 선발하였다. Conserved region에 존재하면서 computer program에 의해서 선발되어진 PMS1과 2의 primer set가 최종적으로 PCR 분석을 위하여 선정되었다. 이 primer set를 사용한 PCR 분석에서, 1ng부터 10pg까지의 웅성 genomic DNA에서 PCR 산물을 얻을 수 있었으며, 자성의 경우는 어떠한 산물도 찾을 수 없었다. PCR에 이용할 수정란의 시료는 2 세포기의 수정란에서 얻었으며 순수 분리된 genomic DNA에서 확립된 조건에서 PCR을 수행하였다. 8개의 수정란을 분석한 결과 4개의 웅성과 4개의 자성 수정란을 확인하였다. 이러한 결과는 선정된 primer set가 돼지 수정란의 성을 조기 감별하는데 효율적인 DNA probe로 사용될 수 있다는 것을 암시한다.