• The Korean Journal of Zoology
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• v.20 no.1
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• pp.9-18
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• 1977

RNA was extracted from the eggs of Xenopus laevis to do preliminary experiments before testing the possibility that if RNase resistant RNA molecules exist in the amphibian egg. Chromatography on Sephadex G-100 column indicated 3 peaks consistently. Only high molecular weight RNA species eluted in the first peak were labeled in vitro using $^{3}H$-dimethyl sulfate to eliminate the possible contribution of base paired oligonucleotides from tRNA. By this method, high specific activity could be obtained and the attached methyl groups were quite stable.

• Journal of Microbiology
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• v.41 no.3
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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.

• Journal of the Korean Chemical Society
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• v.65 no.2
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• pp.121-124
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• 2021

RNA molecules which bind to the G-rich sequence in the 5'-UTR RNA which plays an important role in expression of N-ras, were selected. The secondary structures of five selected RNA aptamers including primer sequence were found by the CLC RNA workbench ver. 4.2 program (www.clcbio.com) and investigated with RNA structural probes such as RNase T1 which has specificity for a G in single-stranded region, RNase V1 specific for double strand and nuclease S1 specific for single strand. The generalized secondary structure model was proposed and characterized. It was composed of a central long double strand region flanked by single strand region at both end sides. The double strand region had an internal single-strand region and bulges. The single strand loop in the right side was composed of four or five nucleotides.

• Journal of Plant Biology
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• v.37 no.3
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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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• v.28 no.6
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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.

• The Plant Pathology Journal
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• v.35 no.1
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• pp.41-50
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• 2019

Sugarcane bacilliform viruses (SCBV), which belong to the genus Badnavirus, family Caulimoviridae, are an important DNA virus complex that infects sugarcane. To explore the genetic diversity of the sugarcane-infecting badnavirus complex in China, we tested 392 sugarcane leaf samples collected from Fujian, Yunnan, and Hainan provinces for the occurrence of SCBV by polymerase chain reaction (PCR) assays using published primers SCBV-F and SCBV-R that target the reverse transcriptase/ribonuclease H (RT/RNase H) regions of the viral genome. A total of 111 PCR-amplified fragments (726 bp) from 63 SCBV-positive samples were cloned and sequenced. A neighbor-joining phylogenetic tree was constructed based on the SCBV sequences from this study and 34 published sequences representing 18 different phylogroups or genotypes (SCBV-A to -R). All SCBV-tested isolates could be classified into 20 SCBV phylogenetic groups from SCBV-A to -T. Of nine SCBV phylogroups reported in this study, two novel phylogroups, SCBV-S and SCBV-T, that share 90.0-93.2% sequence identity and show 0.07-0.11 genetic distance with each other in the RT/RNase H region, are proposed. SCBV-S had 57.6-92.2% sequence identity and 0.09-0.66 genetic distance, while SCBV-T had 58.4-90.0% sequence identity and 0.11-0.63 genetic distance compared with the published SCBV phylogroups. Additionally, two other Badnavirus species, Sugarcane bacilliform MO virus (SCBMOV) and Sugarcane bacilliform IM virus (SCBIMV), which originally clustered in phylogenetic groups SCBV-E and SCBV-F, respectively, are first reported in China. Our findings will help to understand the level of genetic heterogeneity present in the complex of Badnavirus species that infect sugarcane.

• Bulletin of the Korean Chemical Society
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• v.26 no.12
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• pp.2033-2037
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• 2005

In vitro selection was used to isolate $Mg^{2+}$-dependent self-cleaving ribozymes with cis-cleavage activity from a pre-tRNA library having 40-mer random sequences attached to 5'-end of E. coli $tRNA^{Phe}$. After 8 rounds of SELEX (Systematic Evolution of Ligands by Exponential Enrichment), RNA molecules which can self-cleave at the high concentration of $Mg^{2+}$ were isolated. The selected ribozymes can carry out the self-cleavage reaction in the presence of 100 mM $Mg^{2+}$ but not in 10 mM $Mg^{2+}$. The cleavage sites of the ribozymes are located at +3 and +4 of $tRNA^{Phe}$, compared with +1 position of 5'-end cleavage site of pre-tRNA by RNase P. New RNA constructs deprived of its D stem-loop, anticodon stem-loop, variable loop and T stem-loop, respectively showed the cleavage specificity identical to a ribozyme having the intact tRNA structure. Also, the new ribozyme fused with both a ribozyme and $tRNA^{Leu}$ showed the cleavage activities at the various sites within its sequences, different from two sites of position +3 and +4 observed in the ribozyme with $tRNA^{Phe}$. Our results suggest that the selected ribozyme is not structural-specific for tRNA.

• BMB Reports
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• v.28 no.6
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• pp.509-514
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• 1995

A putative secondary structure of the mRNA for the human hepatitis B virus (HBV) X gene is proposed based on not only chemical and enzymatic determination of its single- and double-stranded regions but also selection by the computer program MFOLD for energy minimum conformation under the constraints that the experimentally determined nucleotides were forced or prohibited to base pair. An RNA of 536 nucleotides including the 461-nucleotide HBV X mRNA sequence was synthesized in vitro by the phage T7 RNA polymerase transcription. The thermally renatured transcripts were subjected to chemical modifications with dimethylsulfate and kethoxal and enzymatic hydrolysis with single strand-specific RNase T1 and double strand-specific RNase V1, separately. The sites of modification and cleavage were detected by reverse transcriptase extension of 4 different primers. Many nucleotides could be assigned with high confidence, twenty in double-stranded and thirty-seven in Single-stranded regions. These nucleotides were forced and prohibited, respectively, to base pair in running the recursive RNA folding program MFOLD. The results suggest that 6 different regions (5 within X mRNA) of 14~23 nucleotides are Single-stranded. This putative structure provides a good working model and suggests potential target sites for antisense and ribozyme inhibitors and hybridization probes for the HBV X mRNA.

• The Korean Journal of Mycology
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• v.20 no.3
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• pp.234-239
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• 1992

The experiment was performed to find out the possibilities to detect virus infection in oyster mushrooms, Pleurotus species by analysis of doublestranded ribonucleic acid (ds RNA). Ds RNA segments were extracted from virus infected isolates which grew abnormally. But virus free isolates didn't show any ds RNA segments. The ds RNA was consisted of one large segment of 8100 base pairs (bp) and 4 smaller segments with 2170, 2120, 1980 and 1984 bp. Whereas, cell free virus particles showed only one larger ds RNA segment. The ds RNA was dissolved by RNase A in low salt, 0.1 M SSC and melted at $85^{\circ}C$. It was possible to use the ds RNA analysis for detecting virus infection directly from the host cells.

• Korean Journal Plant Pathology
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• v.10 no.3
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• pp.228-234
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• 1994

Genomic RNA was extracted from Cy strain of odontoglossum ringspot tobamovirus (ORSV-Cy) isolated from infected leaves of tobacco cv. Samsun. Size of the genomic RNA was about 6.6 kb in length. The genomic RNA was fractionated using Sephadex G-50 column chromatography into 2 fractions. They were polyadenylated at their 3'-end using E. coli poly(A) polymerase. Polyadenylated viral RNA was recovered by oligo (dT) primer adapter containing NotI restriction site and Moloney murine leukemia virus SuperScript reverse transcriptase (RNase H-). Second-strand cDNA was synthesized by using E. coli DNA ligase, E. coli DNA polymerase I and E. coli RNase H. Recombinant plasmids containing cDNAs for ORSV-Cy RNA ranged from about 800 bp to 3,000 bp. Among the selected 238 recombinants, pORCY-124 clone was the largest one covering 3'-terminal half of the viral RNA. This clone contained two restriction sites for EcoRI and XbaI and one site for AccI, AvaI, BglII, BstXI, HindIII, PstI, and TthIII 1. respectively. The clone contained partial viral replicase, a full-length movement protein and a complete coat protein genes followed by a 3' untranslated region of 414 nucleotides based on restriction mapping and nucleotide sequencing analyses. Clones pORCY-028, -068, -072, -187 and -224 were overlapped with the pORCY-124. Clones pORCY-014 and -095 covered 5' half upstream from the middle region of the viral RNA, which was estimated based on restriction mapping and partial sequence analysis. Constructed cDNA library covered more than 90% of the viral genome.