RNA interference (RNAi) has been considered as an alternative strategy to control agricultural pests whereby double-strandedRNA triggers a potent and specific inhibition of its homologous mRNA. Since small double-stranded RNAs are requiredfor various RNAi applications, there is a need for cost-effective methods for producing large quantities of high-qualitydsRNA. Bacillus thuringiensis produces much insecticidal proteins with expression of their encoding genes being drivenby sporulation-dependent promoters. To develop dsRNA mass-production platform utilizing Bt, the pHT1K-EGFP plasmidvector which has cyt1Aa sporulation-dependent promoter was constructed. The transcriptional level of target gene (EGFP)is higher 113 times than Bt reference gene (ssPE). It was applied to protect honeybee from Sacbrood virus, so targetgene was replaced to SBV-vp1. By ingestion of Bt-derived dsRNA to honeybee shows positive effect on SBV suppression.

Bacillus thuringiensis(B.t.)에서 cry gene은 plasmid DNA상에 존재하고 대상 곤충에 살충활성을 나타내는 내독소단백질 형성에 관여하는 주요 유전자이다.파밤나방(spodoptera exigua)에 높은 살충활성을 보이는 B.t. subsp. aizawai KB098 균주의 cry gene이 위치하는 plasmid DNA를 찾기 위해 curing 방법을 사용하였다. KB098균주는 cry1Aa, cry1Ab, cry1C, cry1D 4개의 cry gene을 가지고 있으며, Plasmid DNA는 7개가 확인되었다. Cry gene을 암호화하는 plasmid DNA를 찾기 위한 curing 방법은 LB배지에 KB098균주를 희석 후, 27℃ 진탕배양기에서 24시간 배양한 뒤 NA배지에 spreading 한 후, 42℃조건으로 48시간 배양하여 단일 colony를 얻었다. 위상차현미경으로 관찰했을 때 내독소단백질을 형성하지 않는 colony를 NA배지에 streaking하여 27℃ 조건으로 4일간 배양하였다. 위상차현미경관찰을 통해 내독소단백질을 형성하지 않는 colony를 선발하였다. Curing과정이 제대로 수행되었는지 확인하기 위해 SDS-PAGE를 통하여 분자량 130kDa의 내독소단백질 band가 형성되지 않음을 확인하였으며, PCR증폭을 통해 acrystalliferous균주가 KB098 균주가 가지고 있는 4개의 cry gene을 가지고 있지 않음을 확인하였다. cry gene을 암호화 하는 plasmid DNA를 찾기 위해 KB098균주와 5개의 acrystalliferous균주와의 plasmid DNA pattern을 비교하였다. acrystalliferous균주에서 결실된 plasmid DNA만을 gel elution 한 후에 PCR을 통해 cry gene의 존재를 확인하였다.

cry gene은 Bacillus thuringienesis(B.t.)에서 대상 곤충에 살충활성을 나타내는 내독소단백질을 형성하는 주요 유전자로 특정 plasmid DNA상에 암호화되어 있 다. B.t. subsp. aizawai KB098 균주는 파밤나방(spodoptera exigua)에 높은 살충 활성을 보이는 균주로 본 연구에서는 이 균주의 cry gene이 위치하는 plasmid DNA를 찾고자 하였다. 본 실험에 이용된 KB098균주는 cry1Aa, cry1Ab, cry1C, cry1D 4개의 cry gene을 가지고 있으며, Plasmid DNA는 7개가 확인되었다. Cry gene을 암호화하는 plasmid DNA를 찾기 위해 acrystalliferous균주가 필요하였으 며, 42℃조건으로 48시간 열처리 후 새로운 배지에 27℃ 조건으로 3일간 재배양 하여 sporulation단계에서 위상차현미경관찰을 통해 내독소단백질을 형성하지 않 는 colony를 autolysis 단계까지 배양한 후에 위상차현미경으로 재확인하여 확보 할 수 있었다. 획득한 14개의 acrystalliferous를 균주 중, 서로 다른 pattern의 plasmid DNA를 갖는 5개의 균주를 선발하였고, acrystalliferous재확인을 하기 위 해 SDS-PAGE를 통하여 내독소단백질의 분자량인 130kDa의 band가 형성되지 않음을 확인하였으며, PCR증폭을 통해 acrystalliferous균주가 KB098 균주가 가 지고 있는 4개의 cry gene을 가지고 있지 않음을 확인하였다. cry gene을 암호화 하는 plasmid DNA를 찾기 위해 KB098균주와 선발한 5개의 균주와의 plasmid DNA pattern을 비교한 결과, 두 개의 plasmid DNA band에서의 차이가 확인되 었다.

Bacillus thuringiensis 1-3 (Bt 1-3) which was isolated from a Korean soil sample showed high insecticidal activity against Aedes aegypti as well as Plutella xylostella. The isolate was determined to belong to ssp. aizawai (H7) type by an H antiserum agglutination test and produced bipyramidal-shaped crystal proteins with a molecular weight of 130 kDa. PCR analysis with cry gene specific primers showed that Bt 1-3 contained cry1Aa, cry1Ab, cry1C, cry1D and cry2A gene, differing from spp. aizawai (reference strain) which contains cry1Aa, cry1Ab, cry1C and cry1D. We modified the plasmid capture system (PCS) to clone plasmid from Bt 1-3 through in vitro transposition. Fifty-three clones were acquired and their sizes were approximately 10 kb. Based on the sequence analysis, they were classified according to similarities with four known Bt plasmids, pGI3, pBMB175, pGI1 and pGI2, respectively. One of pGI3-like clones, named as pBt1-3, was fully sequenced and its 20 putative open reading frames (ORFs), Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified. The structure of pBt1-3 showed high similarity with pGI3 which is one of rolling-circle replication (RCR) group VI family.

Bacillus thuringiensis 1-3 (Bt 1-3), belonging to subsp. aizawai (H7), showed different characteristics in plasmid profiles from type strain and had cry2A gene in addition to cry1Aa, cry1Ab, cry1C and cry1D. To clone its plasmids and construct E.coli-Bt shuttle vector, we constructed the plasmid capture system (PCS) by inserting attB sites including lacZ between transposable elements (designated as pPCS-Troy). Through in vitro transposition with total plasmids DNA of Bt 1-3, 53 clones were acquired and their sizes were approximately 10 kb. Based on the sequence analysis, they were classified in four groups showing similarities with four known Bt plasmids, pGI3, pBMB175, pGI1 and pGI2, respectively. One of pGI3-like clones, named as pBt1-3, was fully sequenced and its putative open reading frames (ORFs), Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified. The structure of pBt1-3 showed high similarity with pGI3 which is one of rolling-circle replication (RCR) group VI family. As a donor for construction of shuttle vector, pDonr-attPEm vector harboring erythromycin resistant gene between attP sites was constructed. Through BP recombination with pPCS-Troy-cloned Bt plasmids and pDonr-attPEm, erythromycin resistant gene was transposed to Bt plasmids. This scheme proposes that in vitro transposition using pPCS-Troy and BP recombination using pDonr-attPEm can easily clone Bt plasmids and construct novel shuttle vectors.

The baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV), a large circular double-stranded DNA virus whose genome encodes at least 155 open reading frames (ORFs), is highly pathogenic to a number of lepidopteran insects and widely used to transduce various cells for exogenous gene expression. Although many genes of AcMNPV have been identified, the genome-wide study related to viral replication has not been well announced. In this study, to elucidate DNA replication cascade of AcMNPV, we firstly developed a novel baculovirus genome that can be maintained in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. This genome, named bAc-MK, contains a mini-F replicon and a kanamycin resistance marker. Using a convenient Tn7 transposon-based system, pPCS-S, which contains an ampicillin resistance gene, ORF knock-out mutants were generated by random insertion into bAc-MK genome. These mutants will be suffered DNA microarray to elucidate AcMNPV replication cascade.

Plasmid capture systems (PCS) facilitate cloning and manipulation of circular double-stranded DNA. We recently developed an improved PCS (PCS-LZ) to clone relatively large DNA molecules of 30-150 kb. The PCS-LZ donor consists of a mini-F replicon and a kanamycin resistance marker between Tn7 left and Tn7 right ends. Both the replicon and marker gene of the PCS-LZ donor are transferred into target plasmid DNAs by in vitro transposition, followed by replication in E. coli. Colonies are tested for lacZ expression by blue/white screening. Circular DNAs were obtained from plasmids of Bacillus thuringiensis, genome segments of Cotesia glomerata bracovirus and polymorphic genomes of Autographa californica nucleopolyhedrovirus. PCS-LZ is a powerful tool for use in genomic analysis and mutagenesis in invertebrate pathology, and we are extending its application to include vertebrate research.

Bacillus thuringiensis, an entomopathogenic bacterium belonging to the B. cereus group, harbors numerous extra-chromosomal DNA molecules whose sizes range from 2 to 250 kb. In this study, we used a plasmid capture system (PCS) to clone three small plasmids from B. thuringiensis subsp. kurstaki K1 using PCS which were not found in B. thuringiensis subsp. kurstaki HD-1, and determined the complete nucleotide sequence of plasmid pK1S-1 (5.5 kb). Of the six putative open reading frames (ORF2-ORF7) in pK1S-1, ORF2 (MobK1) showed approximately 90% aa identity with the Mob-proteins of pGI2 and pTX14-2, which are rolling circle replicating group VII (RCR group VII) plasmids from B. thuringiensis. In addition, a putative origin of transfer (oriT) showed 95.8% identity with those of pGI2 and pTX14-2. ORF3 (RepK1) showed relatively low aa identity (17.8-25.2%) with the Rep protein coded by RCR plasmids, however. The putative double-strand origin of replication (dso) and single-strand origin of replication (sso) of pK1S-1 exhibited approximately 70% and 64% identities with those of pGI2 and pTX14-2. ORF6 and 7 showed greater than 50% similarities with alkaline serine protease, which belongs to the subtilase family. The other 2 ORFs were identified as hypothetical proteins. To determine the replicon of pK1S-1, seven subclones were contructed in the B. t huringiensis ori-negative pHT1K vector and were electroporated into a plasmid cured B. thuringiensis strain. The 1.6 kb region that included the putative ORF3 (Rep1K), dso and ORF4, exhibited replication ability. These findings identified pK1S-1 as a new RCR group VII plasmid, and determined its replication region.

Bacillus thuringiensis 1-3 (Bt 1-3), belonging to subsp. aizawai (H7), showed different characteristics in plasmid profiles and had cry2A gene in addition to cry1Aa, cry1Ab, cry1C and cry1D. This strain exhibited dual insecticidal activity against Aedes aegypti as well as Plutella xylostella. Recently, we improved the donor-s of plasmid capture system (PCS) by inserting attB sites including lacZ between transposable elements (designated as pPCS-Troy), to construct E.coli-Bt shuttle vector. Through in vitro transposition with total plasmids DNA of Bt 1-3, 53 clones were acquired and their range of sizes were approximately 10 kb. Based on the sequence analysis, they were classified in 4 groups showing similarity with 4 known plasmids, pGI1, pGI2, pGI3 and pBMB175, respectively. One of pGI3-like clones was fully sequenced and its open reading frames were analyzed. As a donor for construction of shuttle vector, pDonr-attPEm vector harboring erythromycin resistant gene between attP sites was constructed. Through BP recombination with pPCS-Troy-cloned Bt plasmids and pDonr-attPEm, erythromycin resistant gene was transposed to Bt plasmids. This scheme proposes that in vitro transposition using pPCS-Troy and BP recombination using pDonr-attPEm can easily construct novel shuttle vectors with any Bt plasmids and this combined procedure can introduce foreign gengs into various circular DNA molecular.

Plasmid capture system (PCS) was developed for easy cloning and manipulation of circular double-stranded DNA from various sources. Recently, we improved PCS system (named PCS-LZ) to clone relatively large-sized DNA molecules (30-150 kb). PCS-LZ donor consists of a Mini-F replicon and a kanamycin resistance marker between Tn7L and Tn7R regions. Both replicon and marker gene of PCS-LZ donor are transferred into target plasmid DNAs by in vitro transposition and the transposed DNAs can replicate in E. coli cells by transformation. White/blue screening by LacZ expression is also available to avoid backgrounds. Up to now, we acquired various circular DNA clones from four sources such as plasmids of B. thuringiensis, bacteriophage genome isolated from B. thuringiensis, genome segments of Cotesia glomerata bracovirus, and polymorphic genomes of Autographa californica nucleopolyhedrovirus. Among them, interestingly, the genome clones of bacteriphage (Ph1-3) were screened from the PCS transposition with plasmids of B. thuringiensis 1-3 strain. The genome of Ph1-3 was fully sequenced (46517 bp) and open reading frames were analyzed. In accordance with this genome finding, the phage particles and its DNA were confirmed from the supernatant of B. thuringiensis 1-3. Ph1-3 showed infectivity to B. thuringiensis type strains such as subsp. galleriae, entomocidus, and morrisoni. Based on these results, we screened the existence of phage in B. thuringiensis type strains by PCR with terminase small subunit-specific primers. Ten of 67 type strains showed PCR products and their sequence similarity was more than 70%. Conclusively, we expect this PCS-LZ system would be a powerful tool for genomic analysis and mutagenesis study at the area of invertebrate pathology and further its application will be enlarged to the vertebrate pathology area.

DNA sequencer는 template로 이용하는 DNA의 quality와 sequencing 반응 산물의 정제 방법, 그리고 gel 농도에 민감하다고 알려져 있다. 이에 우리는 plasmid DNA의 준비, 정제, sequencing 반응, gel 농도와 injection medium 등에 대한 최적 조건을 구축하기 위한 연구를 수행하였다. Plasmid DNA 준비과정에서 phenol을 사용한 것 보다 chloroform을 사용한 것이 평균 reading length가 532 bp에서 684 bp로 향상 되었으며, 2.5% DMSO를 첨가한 것이 첨가하지 않은 것에 비해 200 bp 더 길게 염기서열 분석이 되었다. 또한, sequencing 반응산물 정제 시 50 mM EDTA와 0.6 M sodium acetate를 미리 섞어서 pH 8.0으로 맞춘 것을 사용한 것이 50 mM EDTA(pH 8.0)와 0.6 M sodium acetate(pH 5.2)를 각각 사용한 것 보다 20 bp 길게 염기서열 분석이 되었다. Injection medium으로는 실험실에서 resin으로 탈 이온화 시킨 formamide보다 정제된 ABI formamide를 사용한 것이 보다 재현성 있게 reading length가 90 bp 더 길게 분석 되었으며, 4% PAGE gel 보다 3.6% PAGE gel을 사용한 것이 150 bp 더 길게 분석 되었다. Template 준비 시 chloroform으로 정제하고 2.5% DMSO를 첨가, sequencing 반응산물 정제 시 carrier의 pH를 8.0으로 맞춘 것을 이용, 그리고 ABI formamide와 3.6% gel 농도를 사용하는 최적의 조건으로 평균 700 bp, 85% score를 얻을 수 있었다.

Bacillus thuringiensis 1-3 (Bt 1-3), isolated from Korean soil sample, showed high insecticidal activity against Plutella xylostella. Recently, we improved plasmid capture system donor-s (PCS-S) by inserting attB sites including lacZ between transposable elements (designated as pTroy), to reduce background and construct E. coli-Bt shuttle vector. Through in vitro transposition with total plasmid DNA of Bt 1-3, at least 6 different size plasmids of Bt 1-3 were cloned. Among them, 47 clones which have approximately 10 kb plasmid in size were sequenced and 5 contigs were assembled. These contigs showed partial similarity with two known plasmids, pGI3 or pBMB175, separately. These cloned plasmids will acquire erythromycin resistance by BP recombination reaction with pDonrattPEm vector. After transformation into Bt cells, final erythromycin resistant Bt cell might contain novel E. coli-Bt shuttle vector. This scheme proposes that pTroy and pDonr-attPEm system can easily construct new shuttle vector by in vitro transposition, BP reaction, and erythromycin selection with any Bt plasmids.

In order to investigate the MDR gene by plasmid profile, multiplex-PCR and PFGE analysis, it was examined 20 Salmonella Typhimurium isolated from patients stool from 1999 to 2002. The plasmid profile analysis was shown one to five kinds of plasmid in 600bp~150Kb sized. Especially, PT104 isolates obtained has 3 plasmid identically. According to the result of multiplex-PCR, all PT104 isolates amplified only tet(G) and pse1, but aadA2 was not specialized. PFGE analysis of the fragments restricted by XbaⅠ evidently discriminated plasmid DNA among phage types.

6개(個) 품종(品種)의 대두(大豆)로 부터 분리(分離)된 대두(大豆) 근류균(根瘤菌)의 생리적(生理的) 특성(特性) 및 plasmid DNA의 분리(分離)를 조사(調査)한 결과(結果)는 다음과 같다. YEM 배지(培地)에서 S117, S118, 011, DY-1균주(菌株)는 생육속도(生育速度)가 느리고 alkaline 반응(反應)을 나타내었으며, S110, S111, S114, S115, S116, S120, 010균주(菌株)들은 생육속도(生育速度)가 빠르고 산반응(酸反應)을 나타내었다. fast-growing형(型)과 slow-growing형(型) R. japonicum은 모두 극모성 또는 주모성 편모(鞭毛)를 가진 그람 음성(陰性) 간균(桿菌)이며, 한천 평판에서 점액성(粘液性)이 있는 유백색(乳白色) colony를 형성(形成) 하였다. 접종(接種) 7일후(後), fast-growing형(型)의 colony는 직경(直徑)이 2.0-4.0mm였고, slow-growing 형(型)의 colony는 대체로 0.5-1.5였다. fast-growing형(型)은 pH4.5에서 한결같이 감수성(感受性)이 있고, pH9.5에 내성(耐性)이 있는 반면 slow-growing형(型)은 정반대로 나타났다. 조사(調査)된 균주(菌株)들은 모두 생육(生育)을 위한 탄소원(炭素源)으로써 glucose를 이용(利用)하였으며, 010과 321을 제외하고 모든 균주는 starch는 전혀 이용(利用)하지 못하였으며 fast-growing형(型) 만이 sucrose를 이용(利用)하였다. 조사(調査)된 slow-growing R. japonicum은 일반적으로 15-250kb 정도(程度)의 1-3개(個)의 plasmid DNA를 함유하는 반면, fast-growing R. japonicum은 20-250kb정도(程度)의 1-3개(個)의 plamid DNA를 함유(含有)하고 있었다.