• Journal of Microbiology
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• v.33 no.2
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• pp.154-159
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• 1995

SH-14, a novel killer strain of Ustilago maydis was isolated in Korea. It has been reported in other papers that the toxin specificity and double-stranded RNA pattern of SH-14 strain were different from other laboratory strains. In this paper, we analyzed the biochemical characteristics of U. maydis SH-14 virus. Three distinctive peaks were isolated from CsCl density gradient, designated as top (T), intermediate (I) and bottom (B) components. We found that the densities of each components, 1.285, 1.408 g/cm$\^$3/, respectively, are very similar to those of other strains. As previously reported by the analysis of dsRNA in each component, the dsRNA segments are separately encapsidated. Capsid protein of SH-14 virus consists of two proteins about 70 Kd shown by SDS-PAGE analysis. Electron microscopic examination of the virus particles revealed that UmV particles are very similar in size and morphology to all isolates as well as all lab-strains. In order to test immunological cross reactivity of UmV, werstern bolt analysis was carriedout with antiserum against A8 virus. All capsid protein had positive reaction against A8 antibody which indicated that UmV are immunologically cross-reactive with all isolates from Korea. The results presented in this paper may show that UmV isolated from SH-14 strain has very similar biochemical characteristics to those of other UmV. However, the difference in the toxin specificity and the molecular weight of toxin protein from the SH-14 strain has us to conclude that U. maydis SH-14 strain is a new killer type.

• Animal cells and systems
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• v.11 no.2
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• pp.99-104
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• 2007

RNA interference (RNAi) is the phenomenon of gene silencing by double-stranded RNA (dsRNA) at transcriptional and post-transcriptional levels in a sequence-specific manner. Reverse genetic approaches using RNA interference (RNAi) have become a major tool for biological researches since its discovery in the nematode Caenorhabditis elegans. In this review, we overview how the RNAi phenomenon was discovered and how the underlying mechanism has been elucidated. We also describe and discuss how RNAi experiments can be performed and how RNAi can be used for genetic studies.

• Clinical and Experimental Reproductive Medicine
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• v.34 no.4
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• pp.293-303
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• 2007

Objective: Previously, we identified differentially expressed genes between GV and MII stage mouse oocytes using ACP technology. When we study one of GV selective genes, Obox family, we found Obox4 mRNA expression in ovaries that has been reported as expressed exclusively in testis. Therefore, this study was conducted for characterization and functional analysis for Obox4. Methods: Expression of Obox4 mRNA was examined in gonads and oocytes by RT-PCR. To determine the role of Obox4 in oocyte maturation, Obox4 dsRNA was microinjected into the cytoplasm of GV oocytes followed by 16 h of incubation in the plain medium or by 24 h of incubation in the medium containing IBMX. After RNAi, phenotypes and maturation rates were observed, change in mRNA expression was evaluated, and chromosomal status was confirmed by orcein staining. Results: Obox4 has minimal expression in the ovary compared to that of the other family members. When oocytes were cultured for 16 h in M16 medium after RNAi, maturation rate was not changed significantly, compared with that of non-injected or buffer-injected control oocytes. Surprisingly, however, when oocytes were cultured for 24 h in M16 containing IBMX, in which oocytes were supposed to arrest at GV stage, Obox4 RNAi oocytes were advanced to MI and MII. Spindle structure was disappeared and the chromosomes were condensed in the oocytes after Obox4 RNAi. Conclusions: This is the first report on the expression of Obox4 in the ovary and oocytes. Results of the study suggest that Obox4 plays a crucial role in spindle formation and chromosome segregation during meiosis in oocytes. In addition, Obox4 may play an important role in cAMP-dependent signal cascades of GV-arrest in mouse oocytes.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.6
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• pp.781-787
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• 2017

Objective: The early growth response (Egr) family consists of four members (Egr1, Egr2, Egr3, and Egr4) that are zinc finger transcription factors. Among them, Egr3 is involved in transcriptional regulation of target genes during muscle spindle formation and neurite outgrowth. We previously showed that the immunoreactive Egr3 is localized on oocyte spindle and accumulate near the microtubule organizing center during meiosis I in mice. Egr3 was also shown to be localized on spermatocytes. We herein investigated if Egr3 is expressed in mouse gonads and if Egr3 blockade results in any defect in oocyte maturation. Methods: Expression of Egr3 in mouse gonads was examined by reverse transcription-polymerase chain reaction. Full-length Egr3 and truncated Egr3 (${\Delta}Egr3$) complementary RNAs (cRNAs) with Xpress tag at N-terminus and DsRed2 at C-terminus, and small interfering RNA (siRNA) targeting Egr3 were microinjected into mouse oocytes at germinal vesicle stage. Localization of microinjected Egr3 was examined by confocal live imaging and immunofluorescence staining. Results: Egr3 mRNA was detected in mouse ovaries and testes from 1 to 4 week-old mice. An uncharacterized longer transcript containing 5'untranslated region was also detected in 3 and 4 week-old gonads. Microinjected Xpress-Egr3-DsRed2 or Xpress-${\Delta}Egr3$-DsRed2 localized to nuclei and chromosomes during meiotic progression. Microinjection of these cRNAs or Egr3 siRNA in oocytes did not affect meiotic maturation. Immunofluorescence staining of Egr3 in Xpress-${\Delta}Egr3$-DsRed2-injected oocytes showed a positive signal only on meiotic spindle, suggesting that this antibody does not detect endogenous or exogenous Egr3 in mouse oocytes. Conclusion: The results show that Egr3 localizes to chromosomes during meiotic progression and that certain antibodies may not faithfully represent localization of target proteins in oocytes. Egr3 seems to be dispensable during oocyte maturation in mice.

• Research in Plant Disease
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• v.17 no.2
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• pp.211-215
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• 2011

Cucumber mosaic virus (CMV)-like isolate was collected from Raphanus sativus (cv. Choon-hyang), which showed mosaic symptoms. The isolate was confirmed to a strain of CMV by host responses in Vigna unguiculata, Chenopodium amaranticolor and Gomphrena globosa, by viral genome composition with RT-PCR and PCR-RFLP, and by serological analysis. Symptom developed by the strain of CMV was severe in Nicotiana benthamiana, N. glutinosa, N. tabacum (cv. Samsun, cv. Xanthi), Cucumis melo (cv. Early hanover), Cucumis sativus (cv. White wonder), Capsicum annuum (cv. Chung-yang and cv. Geum-top), but mild symptom was developed in Raphanus sativus (cv. Choon-hyang), Brassica rapa ssp. pekinensis (cv. Bul-Am No. 3), and B. juncea (cv. Daenong Jukgot). Newly isolated strain of CMV could infect diverse crops including Solanaceae, Cucurbitaceae and Brassicaceae. We designated the new strain of CMV as Gn-CMV based on the novel infectivity of Brassicaceae. In double-stranded (ds) RNA analysis, Gn-CMV consisted of 3.3, 3.0, and 2.2 kb genomes likewise other strains of CMV. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed 28 kDa of the CMV coat protein. By restriction enzyme mapping using Cac8I, ClaI and MspI of RT-PCR products indicated that Gn-CMV belongs to CMV subgroup I.

• Bulletin of the Korean Chemical Society
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• v.26 no.9
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• pp.1385-1389
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• 2005

Human hepatitis B virus (HBV) is a pathogen related to the development of liver diseases including chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). However, the efficient methods to suppress HBV replication have not been developed yet. Therefore, we have used RNA interference (RNAi) as a potential tool for the suppression of HBV replication. Here, we designed a 21 nt small intefering dsRNA (siRNA) against hepatitis B virus X (HBx) RNA with 3' overhanging ends derived from T7 promoter. It has been reported that HBV X protein plays an important role in HBV gene expression and viral replication. The suppression of HBx gene expression by the 21 nt siRNA was investigated by Northern blot analysis and chloramphenicol acetyl transferase (CAT) assay. The level of HBx mRNA was decreased by siRNA in a dose-dependent manner. We also found that the 21 nt siRNA inhibited the HBV replication in hepatocellular carcinoma cell.

• Development and Reproduction
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• v.11 no.3
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• pp.263-272
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• 2007

Contrast to mouse where its in vitro maturation rates are high without specific supplements or presence of the cumulus cells, there are some species, such as porcine, where its in vitro oocyte maturation rates are still very low. This comparative study was conducted to investigate the role of malate dehydrogenase(Mor2) during oocyte maturation by RNAi in the mouse and porcine. The Mor2 double-stranded RNA(dsRNA) was prepared speciesspecifically and microinjected into the cytoplasm of denuded germinal vesicle(GV) oocytes. Oocytes were cultured for 48 h(porcine) and 16 h(mouse) in M199 with 10% porcine follicular fluid, pyruvate, p-FSH, EGF, cystein, and estradiol-$17{\beta}$. We measured changes in oocyte morphology, maturation rates and mRNA levels after Mor2 RNAi. We confirmed gene sequence-specific knock down of Mor2 mRNA in both species after Mor2 RNAi. In contrast to our previous finding that mMor2 RNAi resulted in GV arrest in the mouse, we found that pMor2 RNAi resulted in MI arrest in denuded porcine oocytes(58%), but developed to MII(84.4%) in COCs. To determine whether this difference between mouse and porcine RNAi is due to differences in culture media, we cultured mouse oocytes in the M199 media for 16 h after mMor2 RNAi. Mouse oocytes were developed to MII stage(62%) and there was no statistical difference compared to that of non-injected(76.8%) and buffer-injected(73.3%) control groups. Therefore, we concluded that the mouse and porcine oocytes are having different metabolic systems in relation to malate dehydrogenase for oocyte maturation. This could be a basis for differences in maturation rates in vitro in two species. Further scrutinized studies on the metabolic pathways would led us in finding better culture system to improve oocyte maturation rates in vitro, especially in more challenging species like the porcine.

• Molecules and Cells
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• v.24 no.2
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• pp.247-254
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• 2007

Improvement in the pharmacokinetic properties of short interfering RNAs (siRNAs) is a prerequisite for the therapeutic application of RNA interference technology. When injected into mice as unmodified siRNAs complexed to DOTAP/Chol-based cationic liposomes, all 12 tested siRNA duplexes caused a strong induction of cytokines including interferon ${\alpha}$, indicating that the immune activation by siRNA duplexes is independent of sequence context. When modified by various combinations of 2'-OMe, 2'-F, and phosphorothioate substitutions, introduction of as little as three 2'-OMe substitutions into the sense strand was sufficient to suppress immune activation by siRNA duplexes, whereas the same modifications were much less efficient at inhibiting the immune response of single stranded siRNAs. It is unlikely that Toll-like receptor 3 (TLR3) signaling is involved in immune stimulation by siRNA/liposome complexes since potent immune activation by ds siRNAs was induced in TLR3 knockout mice. Together, our results indicate that chemical modification of siRNA provides an effective means to avoid unwanted immune activation by therapeutic siRNAs. This improvement in the in vivo properties of siRNAs should greatly facilitate successful development of siRNA therapeutics.

• The Plant Pathology Journal
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• v.22 no.2
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• pp.125-130
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• 2006

An isolate of Peanut stunt virus (PSV) isolated from black locust tree (Robinia pseudo-acacia L.) showing severe mosaic and malformation symptoms, was designated as PSV-Rp. PSV-Rp was characterized by the tests of host range, physical properties, RNA and coat protein composition and RT-PCR analysis. Nucleotide sequences of the cucumoviruses CP genes were also used for identification and differentiation of PSV-Rp. Six plant species were used in the host range test of PSV-Rp. PSV-Rp could be differentiated from each Cucumovirus strain used as a control by symptoms of the plants. The physical properties of PSV-Rp virus were TIP $65^{\circ}C$, DEP $10^{-3}$, and LIP $2{\sim}3$ days. In dsRNA analysis, PSV-Rp consisted of four dsRNAs, but satellite RNA was not detected. Analysis of the coat proteins by SDS-PAGE showed one major protein band of about 31 kDa. RT-PCR using a part of Cucumovirus RNA3 specific primer amplified ${\sim}950bp$ DNA fragments from the crude sap of virus-infected black locust leaves. RFLP analysis of the RT-PCR product could differential PSV-RP from CMV The nucleotide sequence identity between the PSV-Rp CP and the TAV-P CP genes and the PS-V-RP CP and CMV-Y CP genes were 61.6% and 40.5%, respectively. On the other hand, the nucleotide sequence identity of the PSV-Rp CP gene was $70.9%{\sim}73.4%$ in comparison with those of PSV subgroup I (PSV-ER and PSV-J) and 67.3% with that of PSV subgroup II(PSV-W). Especially, the nucleotide sequence identity of PSV-Rp CP gene and that of PSV-Mi that was proposed recently as the type member of a novel PSV subgroup III was 92.4%.

• The Korean Journal of Mycology
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• v.26 no.3 s.86
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• pp.387-395
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• 1998

Mycelial growth, production of microsclerotia and perithecia, and presence of double-stranded RNA were examined in Calonectria ilicicola isolates from several hosts to detect morphological and/or genetic markers for comparison with levels of virulence. Variability in disease severity, production of microsclerotia and perithecia, and mycelial growth was observed across all isolates. None of 35 isolates of C. ilicicola examined contained detectable levels of double-stranded RNA. Disease severity on soybean cultivars correlated positively with production of both microsclerotia and perithecia. Mycelial growth correlated negatively with production of perithecia. Virulence on the cultivars and production of microsclerotia and perithecia were greater in isolates of C. ilicicola from soybean than those from peanut. These results suggest that the ability of isolates to produce microsclerotia and perithecia is a component of inoculum potential. Perithecia production may serve as a useful marker for characterizing virulence or host specialization in this homothallic fungus.