• 한국미생물생명공학회:학술대회논문집
• /
• 한국미생물생명공학회 1986년도 추계학술대회
• /
• pp.529.2-529
• /
• 1986

pSp-Si (1.6kbp) was originally found in pediococcus halophilus to be a cryptic multicopy-plasmid. Hoping that the plasmid can also replicate in Bacillus subtilis, protoplast transformation of strain 207-25 (recE) was performed using pSP-Sl onto which was added the marker of tmrB8 (on 4.9 kbp EcoRI fragment ) or tmrB+ (on 0.9 kbp xbaI fragment) gene. Though the tmrB8 gene can expres tunicamycin-resistance at the single copy state, and the tmrB+ gene exerts the resistance only at the multicopy state, we could not confirm the replication of pSP-Sl (tmrB8) or pSP-Sl(tmrB+) in B. subtilis. During the experiment, however, we unexpectedly found that the circularized 0.9 kbp xgaI fragment (tmrB+) itself, which had no replication origin, could transform strain 207-25 to tunicamycin-resistant by protoplast transformation. Southern hybridization analyses with tmrB+ and other probes revealed the integration of the fragment at a single copy state into a position other than the homologous tmrB gene. This recE independent integration of another tmrB+ gene into the chromosome may contribute to the tunicamycinresistance in the transformants.

• Journal of Microbiology and Biotechnology
• /
• 제16권5호
• /
• pp.724-730
• /
• 2006

A sequence characterized amplified region (SCAR) marker, which was tightly linked with the A1 mating type locus in Phytophthora infestans, was developed. During the random amplified polymorphic DNA-based phylogenic studies of 33 isolates of P infestans collected from year 2002 to 2004, we found an A1 mating type-specific DNA fragment. This 573-bp DNA fragment was generated only in the genomic DNA of the A1 mating types, when OPC-5 primer was used. Based on the specific DNA sequence, we designed the primer sets for generating the A1 mating type-specific 569-bp DNA fragment. When 33 genomic DNAs of P. infestans were subjected to PCR amplification using different primer combinations, the A1 mating type-specific DNA was amplified, when LB-1F and LB-2R primers were used. The specific 569-bp DNA fragment was generated only from all 18 A1 strains, but not from 15 A2 mating type strains. These results corresponded to the mating type discriminating bioassay of 33 isolates of P. infestans. Therefore, the primer combination of LB-1F/LB2R was chosen as a SCAR marker. Overall, this study indicates that the SCAR marker could be developed into a useful tool for mating type determination of P. infestans.

• 한국미생물·생명공학회지
• /
• 제17권2호
• /
• pp.160-164
• /
• 1989

알카리성 Bacillus sp. AL-8의 알카리성 amylase 유전자를 포함하는 5.7Kb의 EcoRI 단편을 pUB 110을 vector로 하여 amylase를 생산하지 못하는 B. subtilis sta-1에서 발현시켰다. 재조합 plasmid pMB802와 pMB809는 숙주세포인 B. subtilis에서 매우 안정하게 유지되었으며 amylase 생산이 공여균 주에서 보다 1.8배 증가하였다. 형질전환주에서 생산된 amylase는 공여균주와 같은 효소적 성질을 나타내었다. 5.7Kb 단편을 E. coli에 subcloning한 결과 3.7Kb의 EcoRI 단편에 알카리성 amylase 유전자가 존재하였다.

• The Plant Pathology Journal
• /
• 제32권3호
• /
• pp.173-181
• /
• 2016

Gene disruption by homologous recombination is widely used to investigate and analyze the function of genes in Fusarium fujikuroi, a fungus that causes bakanae disease and root rot symptoms in rice. To generate gene deletion constructs, the use of conventional cloning methods, which rely on restriction enzymes and ligases, has had limited success due to a lack of unique restriction enzyme sites. Although strategies that avoid the use of restriction enzymes have been employed to overcome this issue, these methods require complicated PCR steps or are frequently inefficient. Here, we introduce a cloning system that utilizes multi-fragment assembly by In-Fusion to generate a gene disruption construct. This method utilizes DNA fragment fusion and requires only one PCR step and one reaction for construction. Using this strategy, a gene disruption construct for Fusarium cyclin C1 (FCC1), which is associated with fumonisin B1 bio-synthesis, was successfully created and used for fungal transformation. In vivo and in vitro experiments using confirmed fcc1 mutants suggest that fumonisin production is closely related to disease symptoms exhibited by F. fujikuroi strain B14. Taken together, this multi-fragment assembly method represents a simpler and a more convenient process for targeted gene disruption in fungi.

• 한국미생물·생명공학회지
• /
• 제13권3호
• /
• pp.297-302
• /
• 1985

미생물중 urease생성능이 아주 강한 B. pasteurii의 Hind III partial digest 된 chromosomal DNA를 E. coli-B. subtilis bifunctional plasmid vector pGR 71으로 E. coli RR1 균주에 cloning 하므로써 그 urease gene을 expression시킬 수 있었다. 그러나 B. subtilis에서는 insertion DNA fragment의 deletion으로 expression되지 않았다. Cloning된 E.coli RR1 균주로부터 분리 정제한 urease gene함유 Plasmid(pGU66)의 restriction map을 작성하여 본 결과 7.1 Mdal의 insertion fragment가 삽입된 12.6Mdal의 plasmid에 Hind III, Bgl II, Xba I, Sal I등 몇 개의 cleavage site 위치를 찾을 수 있었다. Cloning된 E. coli의 urease는 periplasmic space에 많은 비율로 축적되며, 그 효소학적 성질은 donor인 B.pasteurii의 그것과 매우 유사하였다.

• 대한의생명과학회지
• /
• 제8권1호
• /
• pp.39-46
• /
• 2002

Amplified fragment length polymorphism (AFLP) is a recently developed PCR-based high resolution fingerprinting method that is able to generate complex banding patterns which can be used to delineate intraspecific genetic relationships among bacteria. In this study, we have modified and evaluated a PCR-based technique, amplified fragment length polymorphism (AFLP) analysis, for use in fingerprinting strains of Staphylococcus aureus. Single-enzyme amplified fragment length polymorphism (SE-AFLP) analysis was used to perform strain identification of Staphylococus aureus. By careful selection of AFLP primers, it was possible to obtain reproducible and sensitive identification to strain level. AFLP fingerprinting of 5 reference strains of Staphylococcus aureus and 65 strains of Staphylococcus aureus that were isolated from food sources of different area and diverse genomic types of Staphylococcus aureus were recognized. As a result of this study, we found that the AFLP patterns of Staphylococcus aureus isolated from Seoul, Taejeon and Gwang-Ju indicated the close relation with genetic similarity. The main purpose of this study was to find an alternative and reliable fingerprinting method to study the overall genetic diversity, using Staphylococcus aureus species as an example, and observed if the method can be successfully applied to all staphylococcal species.

• 한국환경복원기술학회지
• /
• 제13권5호
• /
• pp.146-153
• /
• 2010

Urban development typically results in many species being confined to small, isolated and degraded habitat fragments. Fragment size and isolation underpin many studies of modified landscape to prevent biodiversity loss. However, habitat characteristics such as vegetation structure and edge effects are less frequently incorporated in planning. The relative influence of biogeographic (e.g. size, isolation) and vegetation parameters on assemblages is poorly understood, but critical for conservation management. In this study, the relative importance of biogeographic and vegetation parameters in explaining the diversity of forest-interior dwelling birds in forest fragments in Chungcheong Province Area. Fragment size and vegetation characteristics were consistently important predictors of bird diversity. Forestinterior bird richness was influenced by fragment size (0.437), wood age (0.332), wood diameter (0.068), and patch shape (-0.079). To preserve bird diversity of Chungcheong Province Area, it is important to consider differing responses of bird diversity to landscape change, move beyond a focus primarily on spatial attributes (size, isolation) to recognize that landscape change also has profound effects on habitat composition and quality. The result is very useful for long-term aspect of biodiversity conservation plan in regional scale.

• 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.

• 미생물학회지
• /
• 제34권1_2호
• /
• pp.58-63
• /
• 1998

'Contig 구성문제'는 random sequencing 단편들로부터 DNA 염기 서열의 정보를 밝혀낼 경우 발생하는 문제이다. 본 연구에서는 이러한 contig 구성문제를 해결하기 위한 알고리즘을 구성하였으며, X-window 응용 프로그램인 XFAP을 개발하였다. XFAP에서는 dimer 빈도 비교 방법을 사용하여 중첩 가능성이 없는 단편을 효과적으로 제거하였다. 이 방법은 단편 쌍 중첩에서 최소 수용 중첩 길이 내의 각 단편 사이의 dimer 빈도 차이를 이용하여 단편 쌍을 선별하는 것이다. 또한 단편 쌍 최대치 정렬 과정의 메모리 사용량을 줄이기 위해서, Myers 알고리즘을 적용하여 linear space에서 최대치 정렬을 구하는 방법을 사용하였다. 그리고 본 프로그램은 사용자들에게 편리한 그래픽 환경을 제공하기 위해서 Motif 라이브러리를 사용하여 X-window에서 구현되었다. 본 프로그램의 테스트 데이터를 생성하기 위해서 GenBank 데이터베이스에서 일정 길이의 염기 서열을 추출한 다음, sequencing시 일어날 수 있는 모든 오류들을 고려하여 단편 샘플을 생성하였다. 단편 샘플에 대해서 dimer 빈도 비교 방법의 효과 및 실행 시간을 측정하였다. 특히 dimer 빈도 비교 방법의 효율은 단편의 길이에 비례하여 증가하는 것으로 나타났다.

• 한국의학물리학회지:의학물리
• /
• 제17권3호
• /
• pp.167-172
• /
• 2006

전산화단층촬영에서 조영 증강을 위한 조영제 주입은 정맥내에 삽입한 카테터를 통해 자동주입기로 주입하고 있다. 정맥내에 위치한 카테터를 제거하는 도중에 IV 카테터가 부러져 정맥내에 남아 있는 카테터의 조각은 환자에게 순환기 질환의 위험을 초래할 수 있다. CT 검사 중에 정맥내에 카테터 조각이 남아있는 2명의 환자를 대상으로 카테터의 크기 및 위치를 정확히 확인하기 위해 MDCT를 이용하여 정맥내 주사부위를 스캔하였다. 3D 재구성은 MPR, MIP, 볼륨렌더링, SSD 등으로 구성하였다. 정맥내에 위치한 카테터 조각을 MDCT로 스캔한 데이터를 3D 재구성으로 정맥내의 위치 및 크기를 확인하였고, 카테터 조각을 제거하는데 일조하였다.