• Journal of Microbiology
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• 제41권3호
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• pp.239-247
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• 2003

The LRV1-4 capsid protein possesses an endoribonuclease activity that is responsible for the single site-specific cleavage in the 5' untranslated region (UTR) of its own viral RNA genome and the formation of a conserved stem-loop structure (stem-loop IV) in the UTR is essential for the accurate RNA cleavage by the capsid protein. To delineate the nucleotide sequences, which are essential for the correct formation of the stem-loop structure for the accurate RNA cleavage by the viral capsid protein, a wildtype minimal RNA transcript (RNA 5' 249-342) and several synthetic RNA transcripts encoding point-mutations in the stem-loop region were generated in an in vitro transcription system, and used as substrates for the RNA cleavage assay and RNase mapping studies. When the RNA 5' 249-342 transcript was subjected to RNase T1 and A mapping studies, the results showed that the predicted RNA secondary structure in the stem-loop region using FOLD analysis only existed in the presence of Mg$\^$2＋/ ions, suggesting that the metal ion stabilizes the stem-loop structure of the substrate RNA in solution. When point-mutated RNA substrates were used in the RNA cleavage assay and RNase T1 mapping study, the specific nucleotide sequences in the stem-loop region were not required for the accurate RNA cleavage by the viral capsid protein, but the formation of a stem-loop like structure in a region (nucleotides from 267 to 287) stabilized by Mg$\^$2＋/ ions was critical for the accurate RNA cleavage. The RNase T1 mapping and EMSA studies revealed that the Ca$\^$2＋/ and Mn$\^$2＋/ ions, among the reagents tested, could change the mobility of the substrate RNA 5' 249-342 on a gel similarly to that of Mg$\^$2＋/ ions, but only Ca$\^$2＋/ ions identically showed the stabilizing effect of Mg$\^$2＋/ ions on the stem-loop structure, suggesting that binding of the metal ions (Mg$\^$2＋/ or Ca$\^$2＋/) onto the RNA substrate in solution causes change and stabilization of the RNA stem-loop structure, and only the substrate RNA with a rigid stem-loop structure in the essential region can be accurately cleaved by the LRV1-4 viral capsid protein.

• 한국안광학회지
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• 제13권4호
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• pp.43-49
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• 2008

목적: 본 연구에서는 UV-A 차단 콘택트렌즈의 UV-A 차단효과를 일반 콘택트렌즈의 효과와 비교하여 보고자 하였다. 방법: RNase A, catalase 및 superoxide dismutase(SOD)를 일반 콘택트렌즈(UV-A 차단율 20%) 및 자외선 차단 콘택트렌즈(UV-A 차단율 85%)로 차단하였을 때의 UV-A에 의한 변성을 아크릴아미드 겔 전기영동법으로 확인하였다. 효소의 용액은 각각 1, 3, 6, 24 및 96시간 동안 365 nm의 UV-A에 노출시켰으며, 콘택트렌즈에 의한 UVA차단 범위는 전안구를 덮는 RGP 렌즈, 소프트 콘택트렌즈, 안경의 크기를 계산하여 각각 50%, 70% 및 100%로 변화를 주었다. 결과: RNase A, catalase 및 SOD의 변성은 UV-A에 노출된 시간이 길어질수록 심하여졌다. 자외선차단 콘택트렌즈의 자외선 차단 효과는 일반 콘택트렌즈와 비교하여 보았을 때 모든 효소에서 차단 효과가 있음을 확인하였다. 콘택트렌즈로 차단된 범위가 줄어들수록 또한 UV-A에 노출된 시간이 길어질수록 자외선 차단 콘택트렌즈의 차단 효과는 감소됨을 알 수 있었다. 결론: 이상의 결과로 미루어 UV-A에 장시간 노출되었을 경우 자외선 차단 콘택트렌즈는 안구 내의 존재하는 효소를 보호하는 효과가 미흡할 것으로 생각되며, 따라서 햇볕 아래에서의 장시간 콘택트렌즈 착용은 충분한 고려를 하여야 할 것으로 제안할 수 있다.

• 대한화학회지
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• 제52권5호
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• pp.580-582
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• 2008

• 한국식물생명공학회:학술대회논문집
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• 한국식물생명공학회 2005년도 추계학술대회 및 한일 식물생명공학 심포지엄
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• pp.193-193
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• 2005

• 한국동물학회:학술대회논문집
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• 한국동물학회 1996년도 한국생물과학협회 국제학술대회
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• pp.233.2-233
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• 1996

No Abstract, See Full Text

• Genomics & Informatics
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• 제3권1호
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• pp.8-14
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• 2005

Although much is known about the molecular biology of platelets, the megakaryocytes' (MKs) molecular biology was not understood so well because of their rareness. By the cloning and characterization of thrombopoietin (TPO), which is the principal regulator of the growth and development of the MKs, researches on the MKs have been growing rapidly. To understand megakaryocytopoiesis, we investigated the gene expression profile of the MKs using oligonucleotide microarray where 10,108 unique genes were spotted. Comparing the fluorescence intensities of which ratio is $\ge$ ${\mid}2{\mid}$, 372 genes were up-regulated and 541 genes were down-regulated in MKs. For confirmatory expression, RNase protection assay (RPA) establishing abundant apoptotic gene expression was carried out. In MKs, many of the known genes, including several platelet related genes, GATA binding protein were highly expressed. Particularly, TGF beta, clusterin (complement lysis inhibitor), and thymosin beta 4 (actin-sequestering molecules) were expressed highly in MKs. As MKs specific expressed genes may regulate normal and pathologic platelet (and/or MK) functions, the transcript profiling using microarray was useful on molecular understanding of MKs,

• BMB Reports
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• 제50권1호
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• pp.20-24
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• 2017

Clustered regularly-interspaced short palindromic repeats (CRISPR) is a new and effective genetic editing tool. CRISPR was initially found in bacteria to protect it from virus invasions. In the first step, specific DNA strands of virus are identified by guide RNA that is composed of crRNA and tracrRNA. Then RNAse III is required for producing crRNA from pre-crRNA. In The second step, a crRNA:tracrRNA:Cas9 complex guides RNase III to cleave target DNA. After cleavage of DNA by CRISPR-Cas9, DNA can be fixed by Non-Homologous End Joining (NHEJ) and Homology Directed Repair (HDR). Whereas NHEJ is simple and random, HDR is much more complex and accurate. Gene editing by CRISPR is able to be applied to various biological field such as agriculture and treating genetic diseases in human.

• 한국축산식품학회지
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• 제31권2호
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• pp.158-165
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• 2011

A sensitive reverse transcriptase-PCR (RT-PCR) method to detect viable Escherichia coli O157:H7 in milk was established. The primer sets were designed based on the nucleotide sequences of the rfbE (per) and wbdN genes in the O157 antigen gene cluster of E. coli O157:H7. RT-PCR using five different primer sets yielded DNA with sizes of 655, 518, 450, and 149-bp, respectively. All five of the E. coli O157:H7 strains were detected by RT-PCR, but 11 other bacterial species were not. The sensitivity of RT-PCR was improved by adding yeast tRNA as a carrier to the crude RNA extract. The RT-PCR amplifying the 149-bp DNA fragment was the most sensitive for detecting E. coli O157:H7 and the most refractory to the bactericidal treatments. Heat treatment at $65^{\circ}C$ for 30 min was the least inhibitory of all bactericidal treatments. Treatment with RNase A strongly inhibited the RT-PCR of heated milk but not unheated milk. This study described RT-PCR methods that are specific and sensitive with a detection limit of 10 E. coli O157:H7 cells, and showed that pre-treating milk samples with RNase A improved the specificity to detect viable bacteria by RT-PCR.

• Journal of Plant Biology
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• 제37권3호
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• pp.349-355
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• 1994

Viral RNA was extracted from a purified orchid strain of tobacco mosaic virus (TMV-O) from Cymbidium "Grace Kelly". Polyadenylated viral RNAs were primed with Not I-oligo (dT) primer-adapter. First-strand cDNAs were reversely transcribed by Moloney murine leukaemia virus reverse transcriptase (RNAse H-), and then second-strand cDNAs were synthesized by RNase H and DNA polymerase I. The resulting double-stranded cDNAs were ligated into pSPORT1 vector and transformed into competent E. coli strain JM109 cells. The size of cDNAs within the recombinant plasmids was ranging from 0.9 to 3.9 kb. Among the selected clones, pTMO-0205 and -0210 covered the 3' half and the 5' half of the viral genomic RNA, respectively, which were covering more than 99% of the viral genemo size based on sequencing analysis. Two cDNA fragments which were 3.1 kb BamHI and NotI fragement released from pTMO-0.205 and 3.3 kb SalI and BamHI fragment released from pTMO-0210 were ligated with T4 DNA ligase. The clone was almost entire length, lacking only 31 nucleotides from the 5' terminus based on the sequencing result. This method was shown to be efficiently applicable to other plant viral gnomic RNA for the construction of cDNA.n of cDNA.

• Bulletin of the Korean Chemical Society
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• 제28권6호
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• pp.1010-1014
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• 2007

M1 RNA is the catalytic component of RNase P, a tRNA-processing enzyme in Escherichia coli. M1 RNA is produced in the cell by transcription of the rnpB gene and subsequent processing at the 3' end. The 3' flanking region of rnpB contains repeated sets of overlapping sequences coding for small proteins. The issue of whether these proteins are expressed remains to be established. In this study, we showed the expression of a small protein encoded by the first repeat within the 3' flanking region of rnpB. Interestingly, protein expression was increased at lower temperatures. The termination efficiency of rnpB terminators was decreased at lower temperatures, suggesting that antitermination is responsible for enhanced protein expression. Moreover, the purified small protein contained M1 RNA, implying a role as a specific RNA-binding protein.