• Journal of Microbiology and Biotechnology
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• 제9권1호
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• pp.91-97
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• 1999

In this work, a 1.9-kb region of enterococcal plasmid p703/5 was isolated and the nucleotide sequence analysis of the region was performed. One major open reading frame (ORF) was identified encoding a polypeptide of 28 kDa. Database comparisons suggested that the protein showed some homology with other bacterial RepA proteins. Upstream of the ORF, a potential dnaA box, AT-rich region and 22-bp tandemly repeated sequences (DNA iterons), a feature typical for many replication ori sites, were recognized. Deletion analysis using Exonuclease III and several restriction enzymes indicated that the three elements and the gene product from the ORF were essential for replication and that the minimum unit of DNA required for replication resided on the 1.2-kb AvaII subfragment. Thus, this gene product was referred to as RepA.

• 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.

• 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.

• Molecules and Cells
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• 제21권3호
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• pp.330-336
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• 2006

Since its identification in 1989, hepatitis C virus has been the subject of extensive research. The biology of the virus and the development of antiviral drugs are closely related. The RNA polymerase activity of nonstructural protein 5B was first demonstrated in 1996. NS5B is believed to localize to the perinuclear region, forming a replicase complex with other viral proteins. It has a typical polymerase structure with thumb, palm, and finger domains encircling the active site. A de novo replication initiation mechanism has been suggested. To date, many small molecule inhibitors are known including nucleoside analogues, non-nucleoside analogues, and pyrophosphate mimics. NS5B interacts with other viral proteins such as core, NS3, 4A, 4B, and 5A. The helicase activity of NS3 seems necessary for RNA strand unwinding during replication, with other nonstructural proteins performing modulatory roles. Cellular proteins interacting with NS5B include VAMP-associated proteins, heIF4AII, hPLIC1, nucleolin, PRK2, ${\alpha}$-actinin, and p68 helicase. The interactions of NS5B with these proteins might play roles in cellular trafficking, signal transduction, and RNA polymerization, as well as the regulation of replication/translation processes.

• 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.

• Food Science and Biotechnology
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• 제18권2호
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• pp.396-401
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• 2009

The nucleotide sequence contains 2 open reading frames encoding a 45-amino-acid protein homologous to a transcriptional repressor protein CopG, and a 203-amino-acid protein homologous to a replication protein RepB. Putative countertranscribed RNA, a double-strand origin, and a single-strand origin were also identified. A shuttle vector, pUCL2.1, for various lactic acid bacteria (LAB) was constructed on the basis of the pCL2.1 replicon, into which an erythromycin-resistance gene as a marker and Escherichia coli ColE1 replication origin were inserted. pUCL2.1 was introduced into E. coli, Lc. lactis, Lactobacillus (Lb.) plantarum, Lb. paraplantarum, and Leuconostoc mesenteroides. The recombinant LAB maintained traits of transformed plasmid in the absence of selection pressure over 40 generations. Therefore, pUCL2.1 could be used as an E. coli/LAB shuttle vector, which is an essential to engineer recombinant LAB strains that are useful for food fermentations.

• 한국미생물·생명공학회지
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• 제20권2호
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• pp.122-128
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• 1992

Runaway replciation plasmid pSY35AT를 이용하여 이 plasmid가 가지고 있는 $cI_{857}$ repressor를 숙주 MC1065를 이용하여 $30^{\circ}C$에서 $37^{\circ}C$로 온도를 올려 shift-up 법으로 생산하였다. $cI_{857}$은 wild type cI repressor 정제법을 수정하여 사용하였으며 정제된 $cI_{857}$ repressor 농도는 0.11mg/ml이었다. 그리고 정제된 $cI_{857}$ repressor와 $^3H-CTP$로 labelinf한 PrOr과의 결합활성은 cell 파쇄액보다 약 23배 높았으며, 온도가 올라갈수록 결합활성은 떨어졌다.

• Archives of Pharmacal Research
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• 제27권1호
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• pp.77-82
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• 2004

DA-125, a novel derivative of adriamycin, is known for its anti-cancer activity. In this study, the inhibitory mechanism of DA-125 on topoisomerase was investigated in the simian virus 40 (SV40) replicating CV-1 cell by studying the SV40 DNA replication intermediates and DNA-topoisomerase complexes. DNA-protein complexes that were formed in the drug-treated cells were quantitated by using a glass filter assay. SV40 DNA replication intermediates that were accumulated in the drug-treated CV-1 cell were analyzed in a high resolution gel. DA-125 did not accumulate B-dimers of SV40 DNA replication intermediates which were found in the adriamycin-treated CV-1 cells. DA-125 induced a dose-dependent formation of the DNA-protein complexes, while adriamycin did not. When adriamycin and etoposide (VP16) were added to the SV40-infected cells at the same time, adriamycin blocked the formation of the DNA-protein complexes induced by VP16 in a dose-dependent manner. However, DA-125 blocked the formation of the DNA-protein complexes induced by VP16 up to the maximum level of the DNA-protein complexes that were induced by DA-125 alone. Adriamycin and DA-125 did not inhibit the formation of the DNA-protein complexes that were caused by camptothecin, a known topoisomerase I poison. DA-125 is bifunctional in inhibiting topoisomerase II because it simultaneously has the properties of the topoisomerase II poison and the DNA intercalator. As a topoisomerase II poison, DA-125 alone induced dose-dependent formation of the DNA-protein complexes. However, as a DNA intercalator, it quantitatively inhibited the formation of the DNA-protein complexes induced by a strong topoisomerase II poison VP16. Furthermore considering that the levels of the DNA-protein complex induced by VP16 were decreased by DA-125 in terms of the topoisomerase II poison, we suggest that DA-125 has a higher affinity to the drug-binding sites of DNA than VP16 has.

• The Plant Pathology Journal
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• 제17권1호
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• pp.22-28
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• 2001

Barley yellow dwarf virus PAV (BYDV-PAV) has a 5.7-kb positive-sense single-stranded RNA genome that contains six open reading frames (ORFs). BYDV-PAV produces three subgenomic RNAs (sgRNAs). The largest of which encodes the coat, 17-kDa, and readthrough proteins from two initiation codons. To investigate the role of intergenic and 3'-proximal noncoding regions (NCRs) in coat protein (CP) expression and BYDV-PAV replication, a full-length infectious cDNA of the RNA genome of an Illinois isolate of BYDV-PAV was constructed downstream of the Cauliflower mosaic virus-35S promoter. Linear DNA molecules of these cDNAs were infectious, expressed the 22-kDa CP, and produced both genomic RNA sgRNAs in ratios similar to those observed in protoplasts inoculated with viral RNA. The portion of 5'NCR of sgRNA1 between ORFs 2 and 3 was not required for, but enhanced translation of CP from ORF3. Mutants containing deletions in the NCR downstream of ORF5 failed to replicate in oat protoplasts. These results indicate that an intact 3$^1$NCR is required for BYDV-PAV replication.

• Journal of Microbiology and Biotechnology
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• 제19권10호
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• pp.1103-1108
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• 2009

The human immunodeficiency virus (HIV)-l protein Tat acts as a transcription transactivator that stimulates expression of the infected viral genome. It is released from infected cells and can similarly affect neighboring cells. The nucleocapsid is an important protein that has a related significant role in early mRNA expression, and which contributes to the rapid viral replication that occurs during HIV-1 infection. To investigate the interaction between the Tat and nucleocapsid proteins, we utilized cDNA micro arrays using pTat and flag NC cotransfection in HEK 293T cells and reverse transcription-polymerase chain reaction to validate the micro array data. Four upregulated genes and nine downregulated genes were selected as candidate genes. Gene ontology analysis was conducted to define the biological process of the input genes. A proteomic approach using PathwayStudio determined the relationship between Tat and nucleocapsid; two automatically built pathways represented the interactions between the upregulated and downregulated genes. The results indicate that the up- and downregulated genes regulate HIV-1 replication and proliferation, and viral entry.