• 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.

• BMB Reports
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• v.46 no.3
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• pp.163-168
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• 2013

The AML1 gene is an essential transcription factor regulating the differentiation of hematopoietic stem cells into mature blood cells. Though at least 12 different alternatively spliced AML1 mRNAs are generated, three splice variants (AML1a, AML1b and AML1c) have been characterized. Here, using the reverse transcription-polymerase chain reaction with outward-facing primers, we identified a novel non-polyadenylated transcript from the AML1 gene, with exons 5 and 6 scrambled. The novel transcript resisted RNase R digestion, indicating it is a circular RNA structure that may originate from products of mRNA alternative splicing. The expression of the novel transcript in different cells or cell lines of human and a number of other species matched those of the canonical transcripts. The discovery provides additional evidence that circular RNA could stably exist in vivo in human, and may also help to understand the mechanism of the regulation of the AML1 gene transcription.

• 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.

• Applied Biological Chemistry
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• v.21 no.2
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• pp.84-102
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• 1978

Barley embryoless half seeds were incubated in medium containing $10{\mu}M$ GA. Time course activity changes of ${\alpha}-amylase$ were studied in extract and medium seperately by the addition of $0.1{\mu}M,\;5{\mu}M,\;and\;10{\mu}M$ ABA in midcourse incubation of 10 hours after GA treatment. MAK profiles of nucleic acids in embryoless half seeds were compared either with $10{\mu}M$ GA treatment or concomitant treatment with $10{\mu}M$ GA and $10{\mu}M$ ABA after 10 hours incubation, Time course changes of weight increase, chlorophyll, protein and RNA consent in addition to RNase activity were studied in the presence of $10{\mu}M$ GA or $10{\mu}M$ ABA in barley coleoptile sections. After 20 hours incubation in the presence of plant hormones, MAK profiles of nucleic acids and reactive distribution of polysome and monosome were investigated. The above results were summarized as follows. 1) The production of ${\alpha}-amylase$ by treatment with GA alone increased at a linear rate in the incubation period and the active secretion of ${\alpha}-amylase$ began from 18 hours incubation in embryoless half seeds. 2) On the contrary to the partial inhibition by addition of $0.1{\mu}M$ ABA, the production of ${\alpha}-amylase$ was completely inhibited by both $5{\mu}M$ and $10{\mu}M$ ABA within 4 hours. Regardless of concentration of GA, the addition of $5{\mu}M$ ABA in midcourse completely inhibited the production of ${\alpha}-amylase$ 3) ABA treatment gave no effect on the secretion of ${\alpha}-amylase$. 4) There were no differences in RNA fractions between GA treatment and concomitant treatment with GA and ABA in the barlye embryoless half seeds. 5) While GA treatment increased the r-RNA fraction, ABA treatment decreased it and increased the s-RNA fraction in the coleoptile sections. 6) GA treatment increased RNA-DNA fraction best ABA treatment decreased it in the coleoptile sections. 7) While GA treatment suppressed RNase activity, ABA treatment increased it in the coleoptile sections. 8) GA treatment gave no great effect on the total RNA but ABA treatment remarkably diminished it in the coleoptile sections. 9) While GA treatment increased the growth and chlorophyll content, ABA treatment decreased them in the coleoptile sections. 10) GA treatment increased the protein synthesis and polysome formation but ABA treatment decreased them in the coleoptile sections. 11) The inhibition effect of ABA on polysome formation seemed to be resulted from the inhibition of r-RNA synthesis by ABA.

• Genomics & Informatics
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• v.13 no.3
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• pp.70-75
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• 2015

MicroRNAs (miRNAs) have been demonstrated to play an important role in carcinogenesis. Previous studies revealed that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity. In this study, we measured the plasma expression levels of three miRNAs (miR-21, miR-27a, and miR-155) to investigate the usefulness of miRNAs for gastric cancer detection. We initially examined plasma miRNA expression levels in a screening cohort consisting of 15 patients with gastric cancer and 15 healthy controls from Korean population, using TaqMan quantitative real-time polymerase chain reaction. We observed that the expression level of miR-27a was significantly higher in patients with gastric cancer than in healthy controls, whereas the miR-21 and miR-155a expression levels were not significantly higher in the patients with gastric cancer. Therefore, we further validated the miR-27a expression level in 73 paired gastric cancer tissues and in a validation plasma cohort from 35 patients with gastric cancer and 35 healthy controls. In both the gastric cancer tissues and the validation plasma cohort, the miR-27a expression levels were significantly higher in patients with gastric cancer. Receiver-operator characteristic (ROC) analysis of the validation cohort, revealed an area under the ROC curve value of 0.70 with 75% sensitivity and 56% specificity in discriminating gastric cancer. Thus, the miR-27a expression level in plasma could be a useful biomarker for the diagnosis and/or prognosis of gastric cancer.

• BMB Reports
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• v.30 no.4
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• pp.258-261
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• 1997

A simple quantitative assay method for ribonuclease activity has been developed. This method is based on the decrease of fluorescence intensity emitted by the ethidium bromide bound to RNA due to the degradation of RNA by ribonuclease. The substrate RNA was reacted with ribonuclease A and the fluorescence intensity was measured after the addition of ethidium bromide. The intensity difference was calculated using a blank reaction mixture containing no RNase. Whole cellular RNA substrate produced a significant error and was not suitable for this assay method possibly because of local microheterogeniety caused by high molecular weight rRNA. but satisfying results were obtained with tRNA substrate. The intensity difference increased linearly by raising enzyme concentration up to $2{\times}10^{-4}$ Kunitz Units of ribonuclease A. More refined and reliable results were obtained by use of initial reaction velocities which were calculated from the plots of intensity difference vs time. A linear relationship between initial velocities and enzyme concentrations was observed up to 0.01 Kunitz Units of enzyme.

• The Korea Journal of Herbology
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• v.21 no.2
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• pp.9-13
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• 2006

Objectives : To determine the DNA sequence of the 18S rRNA gene of the Rehmannia glutinosa and analyze it phylogenetically Methods : Dried root of the Rehmannia glutinosa was ground with a mortar and pestle. Glass beads(0.5 mm in diameter), TE buffer and SDS solution were added to that. The mixture was vortexed vigorously and extracted with the mixture of phenol, chloroform and isoamyl alcohol and with the mixture of the chloroform and isoamyl alcohol. The nucleic acids were precipitated with ethanol and resuspended in TE buffer. Contaminating RNA was digested with RNAse A and the DNA was purified further with the Geneclean Turbo Kit. This DNA was used as a template for amplification of the 18S rRNA gene by PCR. The PCR product was cloned in the pBluescript SK II plasmid by blunt-end ligation and the DNA sequence of the insert was determined. This DNA sequence was analyzed phylogenetically by the BLAST program. Results and Conclusion : Vortexing the ground powder of the dried plant root with glass beads during cell lysis improved recovery of DNA. The DNA sequence of the Rehmannia glutinosa 18S rRNA gene was determined and deposited at the GenBank as the accession number DQ469606. Phylogenetic analysis of that sequence showed the relationship between the members of the family of Scrophulariaceae and also the close relationship of the Buddleja davidii to the members of the Scrophulariaceae family.

• BMB Reports
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• v.40 no.2
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• pp.226-231
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• 2007

Housekeeping genes are widely used as internal controls in a variety of study types, including real time RT-PCR, microarrays, Northern analysis and RNase protection assays. However, even commonly used housekeeping genes may vary in stability depending on the cell type or disease being studied. Thus, it is necessary to identify additional housekeeping-type genes that show sample-independent stability. Here, we used statistical analysis to examine a large human microarray database, seeking genes that were stably expressed in various tissues, disease states and cell lines. We further selected genes that were expressed at different levels, because reference and target genes should be present in similar copy numbers to achieve reliable quantitative results. Real time RT-PCR amplification of three newly identified reference genes, CGI-119, CTBP1 and GOLGAl, alongside three well-known housekeeping genes, B2M, GAPD, and TUBB, confirmed that the newly identified genes were more stably expressed in individual samples with similar ranges. These results collectively suggest that statistical analysis of microarray data can be used to identify new candidate housekeeping genes showing consistent expression across tissues and diseases. Our analysis identified three novel candidate housekeeping genes (CGI-119, GOLGA1, and CTBP1) that could prove useful for normalization across a variety of RNA-based techniques.

• BMB Reports
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• v.30 no.1
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• pp.37-40
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• 1997

Earthworm extracts are known for anti-inflammatory, analgesic. antipyretic, and anticancer effects but can also influence blood circulation. It was previously shown that an earthworm, Lumbricus rubelius. contained a water-extractable anticoagulant which was a heat- and acid-stable molecule with hydrophilic property. In order to uncover the biochemical nature of this molecule, the anticoagulant was processed with various hydrolases such as trypsin, DNase, RNase. and lysozome. When the digested samples were analyzed with an in vitro coagulation test measuring activated partial thromboplastin time (APTT) and agarose gel electrophoresis, the anticoagulant proved to be a relatively homogeneous DNA fragment with relative molecular size around 72 base pairs. Interestingly, the activity was further stimulated with a trypsin digestion. RNA. on the other hand, did not prolong the APTT. It was also demonstrated that the DNA accelerated the antithrombin III (AT-III) inhibition of thrombin from $IC_{50}$ of 0.34 to 0.16 unit determined with S-2238 as a substrate, whereas heparin, a popular anticoagulant. shifted the value to 0.05. Therefore, it is suggested that the DNA could be considered as an alternative antithrombotic agent to heparin, which would exhibits bleeding side effects.

• Molecules and Cells
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• v.19 no.2
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• pp.239-245
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• 2005

Factor for inversion stimulation (FIS), the Escherichia coli protein, is a positive regulator of the transcription of genes that encode stable RNA species, such as rRNA and tRNA. Transcription of the rnpB gene encoding M1 RNA, the catalytic subunit of E. coli RNase P, rapidly declines under stringent conditions, as does that of other stable RNAs. There are multiple putative FIS binding sites upstream of the rnpB promoter. We tested whether FIS binds to these sites, and if so, how it affects rnpB transcription. In vitro binding assays revealed specific binding of FIS to multiple sites in the rnpB promoter region. Interestingly, FIS bound not only to the upstream region of the promoter, but also to the region from +4 to +18. FIS activated rnpB transcription in vitro, but the level of activation was much lower than that of the rrnB promoter for rRNA. We also examined the effects of FIS on rnpB transcription in vivo using isogenic $fis^+$ and $fis^-$ strains. rnpB transcription was higher in the $fis^-$ than the $fis^+$ cells during the transitions from lag to exponential phase, and from exponential to stationary phase.