• 대한수의학회지
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• 제39권4호
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• pp.772-777
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• 1999

The uncaped genomic RNA of bovine viral diarrhea virus (BVDV) initiates translation by recruitment of eukaryotic translation initiation factors at the internal ribosome entry site (IRES). N-terminal protease ($N^{pro}$) is the first translation product of the open reading frame (ORF). By using the vaccinia virus SP6 RNA polymerase transient expression system, we showed previously that deletion of $N^{pro}$ region reduced translation by 21%. To better understand the biological significance of $N^{pro}$ for translation, we carried out a mutational analysis of the $N^{pro}$ region of BVDV cloned in the intercistronic region of a bicistronic reporter plasmid. We constructed a bicistronic expression vector in which the entire 5 UTR and the mutated $N^{pro}$ region (${\Delta}386-901$, ${\Delta}415-901$ and ${\Delta}657-901$) was cloned between two reporter genes, chloramphenicol acetyltransferase (CAT) and luciferase (LUC). In vivo translation analyses showed that $N^{pro}$ region was dispensible for efficient translation. The results indicate that the $N^{pro}$ region is not essential for BVDV RNA translation and the 3' boundary of BVDV IRES is expanded into $N^{pro}$ region, suggesting that $N^{pro}$ may not play a major role in BVDV replication.

• 한국자기공명학회논문지
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• 제20권1호
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• pp.22-26
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• 2016

FANCJ is a DNA helicase which contributes genome stability by resolving G-quadruplex DNA from 5' to 3' direction. In addition to main ATPase helicase core, FANCJ has the protein binding region at its C-terminal part. BRCA1 and BLM are the binding partner of FANCJ and these protein-protein interactions contribute genomic stability and the proper response to replication stress. As the first attempt for studying FANCJ-BLM interaction, we prepared BLM binding region of FANCJ and characterized with CD and NMR spectroscopy. FANCJ (881-941) with N-ter 6xHis was purified as the oligomer. Secondary structure prediction based on CD data revealed that FANCJ (881-941) composed with ${\beta}$ sheet, turn and coils.$^1H-^{15}N$ HSQC spectra showed nonhomogeneous peak intensities with less number of peaks comparing than the number of amino acids in the construct. It indicated that optimization should be necessary for detailed further structural studies.

• Genomics & Informatics
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• 제1권1호
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• pp.55-60
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• 2003

The nucleotide sequence of pZMO1, a small cryptic plasmid of Zymomonas mobilis ATCC10988 was determined. Analysis of 1,680 bp of sequence revealed $69\%$ identity with Shigella sonnei plasmid, pKYM and $61\%$ identity with Nostoc sp. ss DNA replicating plasmid. Analysis of a deduced amino acid sequence of an orf of pZMO1 revealed $75\%$ identity and $90\%$ similarity with the repA gene of Synechocystis sp. plasmid pCA2.4. The upstream region of the repA gene of pZMO1 possesses six directed repeat sequences and two inverted repeat sequences at downstream of the IR consensus sequence of nick region of rolling circle replication (RCR) plasmid. A typical terminator hairpin structure was found at the downstream region of repA gene. Degradation of single-stranded plasmid DNA by S1 nuclease was detected by Southern hybridization. It suggests that pZMO1 replicates by a rolling circle mechanism in Z. mobilis ATCC10988 cells.

• Journal of Microbiology and Biotechnology
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• 제26권8호
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• pp.1473-1480
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• 2016

This study focused on the variations in the non-coding sequences between ctxB and rstR of various CTX phages. The non-coding sequences of CTX-1 and CTX-cla are phage type-specific. The length of the non-coding region of CTX-1 and CTX-cla is 601 and 730 nucleotides, respectively. The non-coding sequence of CTX phage could be divided into three regions. There is a phage type-specific Variable region between two homologous Common regions (Common regions 1 and 2). The non-coding sequence of RS1 element is similar to CTX-1 except that Common region 1 is replaced by a short RS1-specific sequence. The non-coding sequences of CTX-2 and CTX-cla are homologous, indicating the non-coding sequence of CTX-2 is derived from CTX-cla. The non-coding region of CTX-O139 is similar to CTX-cla and CTX-2; however, it contains an extra phage type-specific sequence between Common region 2 and rstR. The variations in the non-coding sequences of CTX phages might be associated with the difference in the replication efficiency and the directionality in the integration into the V. cholerae chromosome.

• 미생물학회지
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• 제38권3호
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• pp.221-229
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• 2002

일본뇌염바이러스(Japanese encephalitis virus, JEV) Nakayama strain을 초기 multiplicity of infection 5.0으로 Vero세포에 감염하여 1년 이상 안정적으로 바이러스를 방출하는 지속감염(persistently-infected)세포주를 확립하였다. 지속감염 세포에서 지속적으로 방출되는 총 11 개의 Plaque 형태 변이바이러스(morphology mutants)클론을 확보하였다. 분리된 변이바이러스의 복제효율을 분석한 결과 생물학적 표현형과 복제효율은 유의하게 상관하였다. 변이바이러스 RNA 게놈 양 말단의 non-coding region 및 envelop 단백질의 ORF에서는 유의한 염기서열 변화가 관찰되지 않아 JEV 약독화에 새로운 인자가 추가로 관여할 가능성을 제시하였다. 변이바이러스에 감염된 신선한 Vero세포는 wild-type JEV의 일반적 감염성상과 다르게 대다수의 세포가 유의할 만한 세포병변현상을 나타내지 않았다. 감염된 Vero세포에서 wild-type JEV 및 large plaque을 형성하는 변이바이러스의 경우 mRNA와 함께 Bcl-2의 발현은 모두 유의하게 감소하였으며 p53은 뚜렷하게 증가하였다. 반면 small plaque을 형성하는 변이바이러스의 감염세포에서는 Bcl-2와 p53 모두 유의한 변화를 볼 수 없었다. 이상의 결과들과 함께, 감염된 Vero세포의 internucleosomal DNA fragmentation과DNA profile의 유형분석에 따르면 궁극적 인 세포병변효과의 변화는 변이바이러스의 복제효율과 더불어 p53에 비의존적인 apoptosis 수위의 전반적인 감소에 기인하는 것으로 판단된다.

• 한국정밀공학회지
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• 제29권11호
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• pp.1256-1263
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• 2012

The present study covers the ultrasonic patterning process to replicate micro-patterns on a polymer substrate. The ultrasonic patterning process uses ultrasonic waves to generate frictional heat between an ultrasonic horn and the polymer substrate, from which the surface region of the polymer substrate is softened sufficiently for the replication of micro-patterns. The ultrasonic patterning process can divided into two categories according to the direction of vibration transmission: direct patterning and indirect patterning. The direct patterning uses a patterned horn, and the ultrasonic vibration is transferred directly from the patterned horn to the substrate. On the contrary, the indirect patterning process uses a plain horn, and the micro-patterns are engraved on a mold that is located below the substrate. Thus, the micro-patterns are replicated as an indirect manner. In this study, these direct and indirect patterning processes are compared in terms of the replication characteristics. Additionally, the possibility of double-side patterning is also discussed in comparison with the conventional single-side patterning process.

• 약학회지
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• 제49권3호
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• pp.217-224
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• 2005

Human Immunodeficiency Virus type 1 (HIV-l) based lentivirus vector has demonstrated great potential as gene therapy vectors mediating efficient gene delivery and long-term transgene expression in both dividing and nondividing cells. However, for clinical studies it must be confirmed that vector preparations are safe and not contaminated by replication competent lentivirus (RCL) related to the parental pathogenic virus, HIV-l. In this study, we would like to establish the method for titration and RCL detection of lentivirus vector. The titration was determined by vector expression containing the green fluorescent protein, GFP in transduced cells. The titer was $1{\times}10^7$ Transducing Unit/ml in the GFP expression assay and $8.9{\times}10^7$ molecules/ml in the real-time PCR. Also, for the detection of RCL, we have used a combination method of PCR and p24 antigen detection. First, PBS/psi and VSV-G region in the genomic DNA of transduced cells was detected by PCR assay. Second, transfer and expression of the HIV-1 gag gene was detected by p24 ELISA. In an attempt to amplify any RCL, the transduced cells were cultured for 3 weeks (amplification phase) and the supernatant of amplified transduced cell was used for the second transduction to determine whether a true RCL was present (indicator phase). Analysis of cells and supernatant at day 6 in indicator phase were negative for PBS/psi, VSV-G, and p24 antigen. These results suggest that they are not mobilized and therefore there are no RCL in amplification phase. Thus, real-time PCR is a reliable and sensitive method for titration and RCL detection of lentivirus vector.

• 한국미생물·생명공학회지
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• 제16권6호
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• pp.476-483
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• 1988

효모 유전자 운반체의 유형을 특징지우는 ARS1 (autonomous replicating sequence), CEN3 (centromere), 2 $\mu$m OR (yeast plasmid의 origin of replication)의 DNA 절편을 재조합하여 만든 유전자 운반체들의 형질전환력, 안정도 및 운반체 수를 효모균주 SHY4(cir$^+$)와 NNY1(cir$^{\circ}$)에서 비교 조사하였다. CEN3를 갖는 유전자 운반체는 매우 안정하나 세포당 수가 하나로 매우 낮으며 2 $\mu$m OR과 ARS1 을 동시에 갖는 유전자 운반체는 안정할 뿐만 아니라 세포당 수도 높으며 균주내에 존재하는 2 $\mu$m 플라스미드는 2 $\mu$m OR을 가지는 유전자 운반체의 복제에 영향을 준다는 사실을 알 수 있었다.

• The Plant Pathology Journal
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• 제28권1호
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• pp.75-80
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• 2012

$Potato$ $virus$ $X$ (PVX) contains $cis$-acting elements including stem-loop 1 (SL1) RNA at the 5' region; SL1 is conserved among all potexviruses. The SL1 at the positive-sense RNA, SL1(+), is required for PVX RNA replication, cell-to-cell movement, and translation. Previous research demonstrated that SL1(+) RNA also serves as the origin of assembly for encapsidation of PVX RNA. To identify the essential sequences and/or regions for capsid protein (CP) subunit recognition within SL1(+) RNA, we used electrophoretic mobility shift assays (EMSA), UV cross-linking, and yeast three-hybrid analyses. The EMSA and UV cross-linking analyses with PVX CP subunits and RNA transcripts corresponding to the SL1(+) RNA showed that the SL1(+) RNA formed complexes with CP subunits. We also conducted EMSA and yeast three-hybrid analyses with RNAs containing various mutations of SL1(+) RNA elements. These analyses indicated that SL1(+) RNA is required for the interaction with PVX CP and that the RNA sequences located at the loop C and tetra loop of the SL1(+) are crucial for CP binding. These results indicate that, in addition to being important for RNA accumulation, the SL1(+) RNA from the 5' region of the PVX genome is also required for specific binding of PVX CP.

• Animal cells and systems
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• 제1권2호
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• pp.363-370
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• 1997

RNase MRP is a ribonucleoprotein complex with a site-specific endonuclease activity. Its original substrate for cleavage is the small mitochondrial RNA near the mitochondrial DNA replication origin, thus it was proposed to generate the primer for mtDNA replication. Recently, it has been shown to have another substrate in the nucleus, such as pre-S.8S ribosomal RNA in nucleolus. The gene for the RNA component of RNase MRP (MRP RNA) was found to be encoded by the nucleus genome, suggesting an interesting intracellular trafficking of MRP RNA to both mitochondria and nucleolus after transcription in nucleus. In this study, genomic DNA encoding MRP RNA was microinjected into the nucleus of Xenopus oocytes, to analyze promoter regions involved in the transcription. It showed that the proximal sequence element and TATA box are important for basal level transcription; octamer motif and Sp1 binding sites are for elevated level transcription. Most of Xenopus MRP RNA was exported out to the cytoplasm following transcription in the nucleus. Utilizing various hybrid constructs, export of MRP RNA was found to be regulated by the promoter and the 5' half of the coding region of the gene. Interestingly, the transcription in nucleus seems to be coupled to the export of MRP RNA to cytoplasm. Intracellular transport of injected MRP RNA can be easily visualized by whole-mount in situ hybridization following microinjection; it also shows possible intra-nuclear sites for transcription and export of MRP RNA.