• 산업기술연구
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• 제28권B호
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• pp.53-57
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• 2008

From the extensive screening for small cryptic plasmid among about 23 lactic acid bacteria (LAB), 2.4 kb of cryptic plasmid was isolated from Lactobacillus farciminis strain KCTC 3681 and named as pLF24. The plasmid pLF24 was a circular molecule of 2,396 base-pairs in length with a G+C content of 38%. Two protein-coding sequences could be predicted. ORF1 and ORF2 showed homologies to plasmids of gram-positive bacteria. The replication protein coded by ORF2 and the plus origin, were similar to replication regions of other gram-positive bacteria as shown in plasmids such as pLH2, pLS141-1 and pLC2. The nucleotide sequence of pLF24 was deposited into Genbank data base with an accession number of EU429343. The newly isolated plasmid can be used for construction of shuttle vector in Lactobacillus bacteria.

• Journal of Microbiology and Biotechnology
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• 제29권8호
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• pp.1273-1280
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• 2019

Edwardsiella piscicida is the causative agent of edwardsiellosis, which has caused enormous economic losses worldwide. In our previous research, an attenuated live vaccine known as WED and based on the virulent strain E. piscicida EIB202 can effectively protect turbots against edwardsiellosis via intraperitoneal injection, while vaccination by immersion exhibits a weaker effect. During the development of the immersion vaccine, we surprisingly found the counts of ${\Delta}pEIB202/EIB202$ colonized on zebrafish were 100 times lower than those of EIB202. However, pEIB202 carries 53 predicted ORFs and has several copies in E. piscicida EIB202, impeding the study of its function. Thus, the replication region is located at a 1,980 bp fragment (from 18,837 to 20,816 bp), containing a transcriptional repressor and a replication protein. Moreover, the minimal replication plasmid, named pRep-q77, has low copies in both E. coli and E. piscicida, but is more stable in E. piscicida than in E. coli. This work lays a foundation for further examination of the function of the virulence plasmid pEIB202.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1994년도 춘계학술대회 and 제3회 신약개발 연구발표회
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• pp.178-178
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• 1994

Hepatitis B Virus (HBV) is a DNA virus with a 3.2kb partially double-stranded genome. The life cycle of the virus involves a reverse transcription of the greater than genome length 3.5kb mRNA. This pegenomic RNA contains all the genetic information encoded by the virus and functions as an intermediate in viral replication. Tumor suppressor p53 has previously been shown to interact with the X-gene product of the HBV, which led us to hypothesize that p53 may act as a negative regulator of HBV replication and the role of the X-gene product is to overcome the p53-mediated restriction. As a first step to prove the above hypothesis, we tested whether p53 represses the propagation of HBV in in vitro replication system. By transient cotransfection of the plasmid containing a complete copy of the HBV genome and/or the plasmid encoding p53, we found that the replication of HBV is specifically blocked by wild-type p53. The levels of HBV DNA, HBs Ag and HBc/e Ag secreted in cell culture media were dramatically reduced upon coexpresion of wild-type p53 but not by the coexpression of the mutants of p53 (G154V and R273L). Furthermore, levels of RNAs originated from HBV genome were repressed more than 10 fold by the cotransfection of the p53 encoding plasmid. These results clearly states that p53 is a nesative regulator of the HBV replication. Next, to addresss the mechanism by which p53 represses the HBV replication, we performed the transient transfection experiments employing the pregenomic/core promoter-CAT(Chloramphenicol Acetyl Transferase) construct as a reporter. Cotransfection of wild-type p53 but not the mutant p53 expression plasmids repressed the CAT activity more than 8 fold. Integrating the above results, we propose that p53 represses the replication of HBV specifically by the down-regulation of the pregenomic/core promoter, which results in the reduced DNA synthesis of HBV. Currently, the mechanism by which HBV overcomes the observed p53-mediated restriction of replication is tinder investigation.

• Journal of Microbiology and Biotechnology
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• 제19권4호
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• pp.403-408
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• 2009

A number of bifidobacterial species of human origin were screened for the presence of cryptic plasmids. One strain, Bifidobacterium longum FI10564, harbored plasmids of approximately 2.2 kb, 3.6 kb, and 4.9 kb in size. The smallest plasmid, pFI2576(2,197 bp), was studied in detail and its complete nucleotide sequence was determined. Computer-assisted analysis of this novel plasmid(G+C content 62%) identified 9 putative open reading frames(orfs), 3 of which were shown to be probable genes. These putative genes are arranged in an operon-like structure, in which the overlapping orfs 1 and 2 encode putative Rep proteins and are highly homologous to the rep genes of the B. longum plasmid pMBI(1,847 bp). The mechanism of replication of pFI2576 was investigated using Southern blot analysis of whole cell lysates, with and without S1 nuclease treatment, and atomic force microscopy(AFM). The results indicate that pFI2576 is likely to use the theta mode of replication.

• Journal of Microbiology
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• 제40권2호
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• pp.140-145
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• 2002

The 5.8 kb pMMH1, rolling-circle replication (RCR) plasmid of the wild type soil Bacillus mesentericus was developed into a novel secretion vector system in Bacillus subtilis. The pMMHl turned out to have a replication origin and two open reading frames (ORFs) of the putative γ-GTP and type I signal peptidase (sipP). To characterize the regions necessary for plasmid stability and high copy number, five vectors (pPS, pPP, pEN, pMN, pME) were constructed by disruption or deletion of each region in pMMH1. Like pMMHl all constructed vectors were stable over 100 generations In a non-selective medium. Since pPS was the smallest (2.3 kb)of all, it was selected for the construction of a navel secretion vector, Using the $\alpha$-amylase promoter/signal sequence of B. subtilils the novel plasmid pJSN was constructed. When $\beta$-glucosidase was expressed using pJSN, we found $\beta$-glucosidase activity in the medium. This result strongly suggested that plasmid pJSN can be used for the production of bioactive peptides in B. subtilis.

• 한국미생물·생명공학회지
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• 제13권4호
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• pp.345-348
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• 1985

Mannitol hypertonic regeneration media를 사용하는 PEG-induced protoplast transformation system을 이용해서 pUB110과 pE194의 recombinant plasmid로 B. subtilis BR151을 transformation 시킴으로써 두 plasmid에서 유래되는 각각의 Neo$^{R}$와 Em$^{R}$을 동일한 recipient cell 내에서 동시에 발현시킬 수 있었다. Neomycin과 erythromycin을 함께 함유하는 mannitol regeneration media상에서 recombinant plasmid의 transformation frequency는 6.5 $\times$ $10^{-5}$이었다. 한편 transformant cell 내에서 recombinant plasmid의 replication이 agarose gel electrophoresis로 확인되었다.

• 신재생에너지
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• 제19권4호
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• pp.46-52
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• 2023

Methane is the second most emitted greenhouse gas after carbon dioxide. Despite lower emissions than those of carbon dioxide, methane receives significant attention owing to its more than 20-fold higher global warming potential. Consequently, the importance of research on methanotrophic bacteria, microorganisms capable of converting methane gas into high-value materials, is increasingly emphasized. In the case of methanotrophic bacteria, knowledge on episomal plasmids that can be used for genetic engineering remains lacking, which poses significant challenges to the engineering process. The replication origin sequences of natural plasmids within methanotrophic bacteria have been predicted through in silico methods. The basic characteristics of the replication origin, such as a high A/T ratio, repetitive sequences, and proximity to proteins related to replication, have been used as criteria for identifying the replication origin. As a result, a region with a sequence of 18 base pairs repeated eight times could be identified. The putative replication origin sequence thus identified generally takes the form of iterons, but it also possesses unique features such as the length of the gap between iterons and the repetition of identical iteron sequences. This information can be valuable for future design of episomal plasmids applicable to methanotrophs.

• BMB Reports
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• 제34권6호
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• pp.584-589
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• 2001

One of the cryptic plasmids from the oil degrading bacterium Klebsiella sp. KCL-2, the small plasmid pGD2, has been identified and characterized. This plasmid has a size of 3.6 kb with unknown functions. We constructed the recombinant plasmid pMGD2. The nucleotide sequences of the plasmid were determined and two open reading frames were detected. ORF1 encodes a replication initiator protein (RepA), which has a high degree of homology with the protein of ColE2 plasmid. The product encoded by ORF2 showed a high similarity with the transposase protein of IS5. IS5 is 1195 by long and contains an inverted terminal repetition of 16 bp with one mismatch. Stem-loop structures in the 5'untranslated region of the repA suggest that a putative gene, incA, is located in a complementary strand to the leader region of the repA mRNA.

• Journal of Microbiology and Biotechnology
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• 제13권6호
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• pp.1018-1020
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• 2003

The complete 1,486 nucleotide sequence of a cryptic plasmid separated from Lactobacillus bifermentans strain A02 isolated from Kimchi has been determined. The plasmid, designated as pA021, encodes a 33,488 Da putative Rep protein. Based on the sequence similarity, the protein shows homology with coding protein of pRS1, a previously reported plasmid of Oenococcus oeni and the replication initiation protein (Rep) of the Staphylococcal pT181 plasmid family.

• Journal of Microbiology and Biotechnology
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• 제13권3호
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• pp.457-462
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• 2003

The complete nucleotide sequence of a plasmid, pMG1, isolated from Bifidobacterium longum MG1 has been determined. This plasmid, composed of 3,862 base pairs with 65.1% of G+C content. harbors two major open reading frames (ORF) encoding putative proteins of 29 kDa (ORF I) and 71 kDa (ORF II). ORF I showed relatively high amino acid sequence homology with replication proteins of other plasmids from Gr Im-positive and -negative bacteria. Upstream of ORF I, four sets of tandem repeat sequences resembling the iteron structure of related plasmids were found. S1 endonuclease treatment and Southern blot analysis revealed that pMG1 accumulates single-stranded DNA (ssDNA) intermediate, which indicate i the rolling circle replication (RCR) mechanism of this plasmid. Homology search indicated that ORF II encodes plasmid mobilization protein, and the presence of highly conserved oriT sequence in the upstream of this gene supported this assumption. RT-PCR showed that only ORF I is expressed in vivo. Based on these results, pMG 1 was exploited to construct a shuttle vector, pBES2. It was successfully transformed into Bifidobacterium and maintained stably.