• Journal of Microbiology and Biotechnology
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• 제3권4호
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• pp.256-260
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• 1993

DNA subfragments, sopA, sopB and sopC which help to maintain the stability of an ori C plasmid, were derived from a mini-F plasmid DNA (EcoRI restriction fragment f5) after digestion with restriction endonuclease, and cloned in the vector plasmid pBR322. The recombinant plasmids obtained were introduced into E. coli KY7231 and E. coli CSR603 strains, and proteins specified by the mini-F fragments were analysed by SDS-PAGE. Two proteins encoded by the F fragments were detected, and their molecular weights were 41,000 and 37,000 daltons. Fluorography after one and two dimensional gel electrophoresis of the lysates showed that these two proteins had been overproduced in the cells which were allowed to incorporate radioactive amino acid after plasmid amplification by chloramphenicol treatment. The isoelectric points of sopA and sopB proteins were 6.6 and 7.0, respectively.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 1986년도 추계학술대회
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• pp.525.3-526
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• 1986

For the analysis of fermentation characteristics and productivity of plasmid coded product, car-boxymethylcellulase in a recombinant DNA cell fermentation system, batch and continuous fermentations were carried out using a Bacillus megaterium ATCC 14945 transformed with a plasmid, pCK 108 haboring carboxymethyl cellulase gene. The effects of carbon and nitrogen sources and of temperature and pH on cell growth, product yield, plasmid stability, specific plasmid contents of cell, and gene expression efficiency were carefully studied. These experimental results will be discussed in some details.

• 미생물학회지
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• 제27권3호
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• pp.188-193
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• 1989

Gene for lactose catabolism in Lactobacillus casei SW-M1 was encoded by a 60Kb metabolic plasmid. A derivative of only 10kb, pPlac 15 of recombinant plasmid, was constructed by introducing into pBR322 and was cloned into E. coli using restriction endonuclease Pst I. A 10kb insery DNA in plasmid pBR322 was identified as a gene encoded phospho-$\beta$-galactosidase by the determination of enzyme activity. Phospho-$\beta$-galactosidase was apparently expressed in E. coli. The enzyme activities of cell-free extract from transformant E. coli HB101 carrying pPLac 15 DNA were not different from that of L. casei as a donor strain on the basis of enzyme properites. However, specific activity of phospho-$\beta$-galactosidase in the cloned strain with Lac $Y^{-}$ phenotype of E. coli HB101 was lower than that in L. casei strain.

• 미생물학회지
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• 제22권4호
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• pp.249-255
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• 1984

To develop the host-vector system for industrial Coryneform bacteria that seemed to be the most suitable microorganisms for molecular breeding of genes involved in the production of amion acids, nucleotides, and other products of industrial interest, broad host range E. coli plasmid R 1162 DNA was transformed into Brevibacterium ammoniagenes and the plasmids pKU6 isolated from a transformant was physically characterized. All other plasmids from the transformed cells except pKU6 exsisted as multimeric forms in Brevibacterium ammoniagenes. The plasmid DNA was retransformed into Corynebacterium glutamicum with a high frequency ($1.32{\times}10^{-1}$ per cell) and maintained stably both in Brevibacterium ammoniagenes and Corynebacterium glutamicum after 100 generations of cultures with 25-30 copy number per cell. The size of both plasmid pKU6 and plasmid R1162 were the same and restriction maps by EcoR I, Ava I, Pst I, Pvu II and Hinc II were also similar.

• 미생물학회지
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• 제30권3호
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• pp.218-224
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• 1992

Carboxydobacteria 의 일산화 탄소 산화에 대한 유전학적 연구를 위해 Pseudomonas caarboxydovorans 에 존재하는 pYK100 plasmid 와 pBR322 를 이용하여 pYK322 (7.2 kb, Ap, Tc) 와 pYK324 (7.2 kb, Ap, Tc) 등 두가지 재조합 plasmid shuttle 백테를 만들고, pYK100와 pACYC184를 이용하여 pYK210(5.2 kb, $CM^{r}$ ), pYK220 (5.2kb,$CM^{r}$ ), pYK230 (5.2 kb, $Cm^{r}$ ), pYK232 (5.2 kb, $CM^{r}$) 등 네가지 shuttle 벡터를 만들었다. 재조합된 벡터들은 보두 대장균에서 안정되게 복제되었다. pYK322 와 pYK220 을 이용한 carboxydobacteria 의 형질전환 실험에서 Bagdasarian 과 Timmis 의 방법 (Curr. Top. Microbiol. Immunol., 96 :47-67, 1982) 을 변형하여 0.2% succinate 가 포함된 무기염류배지에서 지수성장 중기까지 배댜ㄷ한 세균을 이용하고, 형질전환용액의 10 mM RbCI 을 100 mM KCI 로 대체하며, 형질전환용액 처리후 4.deg.C 에서의 방치시간을 12시간으로 하고, DNA첨가휴 45.deg.C 에서 3 분간 heat shock 을 준 경우에 높은 형질전환이 일어났다. 형질전환된 세균으로 부터 형질전환에 사용한 plasmid 를 발견할 수 없었는데, 이는 도입된 plasmid 가 염색체 DNA 에 결합되었기 때문인 것으로 추측된다.

• 미생물학회지
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• 제34권4호
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• pp.236-242
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• 1998

박테리오파지 P2-P4 시스템은 바이러스 조립과정 기작 연구를 위한 뛰어난 실험 소재로 연구되어 왔으나, 이 시스템에 유전자 조작상 필수적인 유용한 플라스미드가 없는 관계로 그의 필요성이 대두되고 있다. 본연구에서는 도움파지가 없을 때 플라스미드 상태로 존재하는 P4 ash8 (sid71)을 시작물질로, 항생제 내성 유전자를 도입하여 선택성을 줄 목적으로 P4 파지 증식에 불필요한 P4 DNA 단편을 pUC4-K 플라스미드의 kmr(kanamycin resistance) 유전자로 치환하여 P4 ash8(sid71) kmr을 생성하였다. 이 플라스미드는 박테리오파지 P2로 induction하여 박테리오파지 P4 상태로 증식시킬 수 있게 그 genome 크기를 조정하였으며, 파지 상태로 전환된 것을 burst size 결정 및 CsCl 부양 균등밀도 편차실험을 통해 입증하였다. 생성된 P4 플라스미드 유도체를 유전자 조작하여 P4의 integrase 기능을 잃은 변이체를 쉽게 만들 수 있었으며, 역시 박테리오파지 P4 상태로 증식시킬 수 있었다. 이러한 변이체 플라스미드의 안정성 실험을 수행하여, P4의 integrase 기능이 지속적인 플라스미드 상태 유지를 위해 필요하다는 것을 보여주었다.

• Journal of Microbiology and Biotechnology
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• 제10권4호
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• pp.535-538
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• 2000

Streptomyces lavendulae FRI-5 produces the ${\gamma}$-butyrolactone autoregulator IM-2, which is required for nucleoside antibiotic producetion. We have developed a system for introducing DNA into S. lavendule FRI-5 via conjugal transfer from Esherichia cole. Conditions were established for conjugation of the oriT-and attP-containing plasmid pSET152 from E. coli ET12567 (pUZ8002) to FRI-5. Conjugation resulted in integration of the plasmid at the chromosomal C31 attB site. The frequency of intergeneric conjugation varied with the medium used. The highest frequency ($1.6\times10-5$ per recipient) was obtained on ISP medium 2 containing 10mM MgCl2. Southern blot and phenotypic analyses of exconjugants revealed that S. lavendulae FRI-5 contains a unique C31 attB site, and that integration of heterologous DNA into the attB site did not interfere with morphological differentiation or IM-2-dependent signal transduction, including the production of a blue pigment. This system will now enable detailed genetic analysis of the regulation of antibiotic production in S. lavendulae FRI-5.

• Asian Pacific Journal of Cancer Prevention
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• 제17권sup3호
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• pp.299-304
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• 2016

Cytolethal distending toxin (CDT) is a secreted tripartite genotoxin produced by many pathogenic gram-negative bacteria. It is composed of three subunits, CdtA, CdtB and CdtC, and CdtB-associated deoxyribonuclease (DNase) activity is essential for the CDT toxicity. In the present study, to design a novel potentially antitumor drug against lung cancer, the possible mechanisms of cdtB anticancer properties were explored in the A549 human lung adenocarcinoma cell line. A recombinant plasmid pcDNA3.1/cdtB was constructed expressing CdtB of human periodontal bacterium Aggregatibacter actinomycetemcomitans and investigated for toxic properties in A549 cells and possible mechanisms. It was observed that plasmid pcDNA3.1/cdtB caused loss of cell viability, morphologic changes and induction of apoptosis. Furthermore, measurement of caspase activity indicated involvement of an intrinsic pathway of cell apoptosis. Consequently, the recombinant plasmid pcDNA3.1/cdtB may have potential as a new class of therapeutic agent for gene therapy of lung cancer.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 1991년도 춘계학술발표대회 논문집
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• pp.237-242
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• 1991

In order to increase the production of glutathione by maximizing the expression of recombinant gsh plasmids, two genes responsible for the biosynthesis of glutathione were cloned. A gshI gene was cloned onto pBR322 plasmid as 3.6Kb PstI DNA fragment from E. coli K-12 chromosomal DNA. Also gshII gene was cloned onto pUC13 plasmid as 2.2Kb PstI-BamHI DNA fragment. In order to improve the glutathione producing activity more efficiently, various recombinant plasmids containing tandem repeated gshI genes or both genes in various copy number onto the same vector were constructed. E. coli cells harboring pGH501 plasmid (pUC8-gshI$\cdot$I$\cdot$II) showed the highest glutathione synthesizing activity. The conditions for glutathione production with an ATP-generating system such as acetate kinase reaction of E. coli cells or glycolytic pathway of yeast cells were examined using the E. coli cells harboring the pGH501 plasmid. When the acetate kinase reaction of E. coli cells was used as an ATP generating system, 20mM of L-csteine was converted into glutathione with a yield of $100\%$.

• Journal of Microbiology
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• 제37권4호
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• pp.229-233
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• 1999

The mitochondrial plasmid pMLP1 from a white-rot fungus, Pleurotus ostreatus, is a double-stranded DNA containing 381 bp terminal inverted repeat (TIR) whose 5'-ends are covalently bound by terminal proteins. The plasmid contains two major open reading frames (ORFs), encoding putative DNA and RNA polymerases, and a minor ORF encoding a small, highly basic protein. To identify the DNA binding activity that recognizes the TIR region of pMLP1, gel retardation assays were performed with mitochondrial extracts. A specific protein binding to a region between 123 and 248 nt within TIR was observed. We examined whether the gene product of mORF1 bindes to this region specifically. E. coli cell extract which contains an overproduced mORF1 protein formed a complex specific to the region between 123 and 248 nt. Inclusion of mORF1 protein in the specific complex formed between P. ostreatus mitochondrial extract and TIR was confirmed by a supershift assay using polyclonal antibodies against the mORF1 protein. Our result suggest that the product of mORF1 may function as a terminal region recognition factor (TRF), recognizing an internal region in TIR.