• 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.

• Animal Systematics, Evolution and Diversity
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• no.nspc3
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• pp.139-146
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• 1992

The primary sequence of the 18S rRNA gene of a crustacean Pugettia quadridens (Decapoda: Pleocyemata: Brachyura) was determined by the PCR cloning and Taq sequencing. The 18S rRNA gene of this species in 1837 bases long, and 46 bases shorter than that of another crustacean decapod Oedignathus inermis. The similarity between two species is 90.8% when the insertion and/or deletion sites were excluded. Within the molecule, the most conservative (identical) region locates at the position of 1137-1206 and it is 70 bases long. The most long consecutive nucleotide differences occur at the position between 46-55 and the second most between 399-407. The sequence variation in the primary structure of 18S rRNA gene are not evenly distributed throughout the molecule.

• Molecules and Cells
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• v.45 no.8
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• pp.588-602
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• 2022

Various RNA-binding proteins (RBPs) are key components in RNA metabolism and contribute to several neurodevelopmental disorders. To date, only a few of such RBPs have been characterized for their roles in neocortex development. Here, we show that the RBP, Rbms1, is required for radial migration, polarization and differentiation of neuronal progenitors to neurons in the neocortex development. Rbms1 expression is highest in the early development in the developing cortex, with its expression gradually diminishing from embryonic day 13.5 (E13.5) to postnatal day 0 (P0). From in utero electroporation (IUE) experiments when Rbms1 levels are knocked down in neuronal progenitors, their transition from multipolar to bipolar state is delayed and this is accompanied by a delay in radial migration of these cells. Reduced Rbms1 levels in vivo also reduces differentiation as evidenced by a decrease in levels of several differentiation markers, meanwhile having no significant effects on proliferation and cell cycle rates of these cells. As an RNA binding protein, we profiled the RNA binders of Rbms1 by a cross-linked-RIP sequencing assay, followed by quantitative real-time polymerase chain reaction verification and showed that Rbms1 binds and stabilizes the mRNA for Efr3a, a signaling adapter protein. We also demonstrate that ectopic Efr3a can recover the cells from the migration defects due to loss of Rbms1, both in vivo and in vitro migration assays with cultured cells. These imply that one of the functions of Rbms1 involves the stabilization of Efr3a RNA message, required for migration and maturation of neuronal progenitors in radial migration in the developing neocortex.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.9
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• pp.3997-4003
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• 2014

Background: Infection with certain human papillomavirus (HPV) genotypes is the most important risk factor related with cervical cancer. The objective of the present study was to investigate the prevalence of HPV infection, the distribution of HPV genotypes and HPV E6/E7 oncogene mRNA expression in Turkish women with different cervical cytological findings in Mersin province, Southern Turkey. Materials and Methods: A total of 476 cytological samples belonging to women with normal and abnormal cervical Pap smears were enrolled in the study. For the detection and genotyping assay, a PCR/direct cycle sequencing approach was used. E6/E7 mRNA expression of HPV-16, 18, 31, 33, and 45 was determined by type-specific real-time NASBA assay (NucliSENS EasyQ$^{(R)}$HPV v1.1). Results: Of the 476 samples, 106 (22.3%) were found to be positive for HPV DNA by PCR. The presence of HPV was significantly more common (p<0.001) in HSIL (6/8, 75%) when compared with LSIL (6/14, 42.9%), ASC-US (22/74, 29.7%) and normal cytology (72/380, 18.9%). The most prevalent genotypes were, in descending order of frequency, HPV genotype 66 (22.6%), 16 (20.8%), 6 (14.2%), 31 (11.3%), 53 (5.7%), and 83 (4.7%). HPV E6/E7 oncogene mRNA positivity (12/476, 2.5%) was lower than DNA positivity (38/476, 7.9%). Conclusions: Our data present a wide distribution of HPV genotypes in the analyzed population. HPV genotypes 66, 16, 6, 31, 53 and 83 were the predominant types and most of them were potential carcinogenic types. Because of the differences between HPV E6/E7 mRNA and DNA positivity, further studies are required to test the role of mRNA testing in the triage of women with abnormal cervical cytology or follow up of HPV DNA positive and cytology negative. These epidemiological data will be important to determine the future impact of vaccination on HPV infected women in our region.

• Journal of fish pathology
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• v.17 no.3
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• pp.159-169
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• 2004

Bacterial wihte enteritis ocurred by infection of V. ichthyoenteri is a devastating disease in olive flounder (Paralichthys olivaceus) hatcheries in Korea. Since white enteritis has been a problem in aquqtic industries, necessity of a rapid detection method is increased. In an attempt to develop rapid PCR method the detection of V. ichthyoenteri, we examined the 16S-23S rRNA intergenic spacer region(ISR) of V. ichthyoenteri and developed species-specific primer for V. ichthyoenteri. The intergenic spacers were amplified by primers complementary to conserved region of 16S and 23S rRNA genes. The intergenic spacer region between the 16S and 23S rRNA genes of V. ichthoenteri were investigated by PCR fragment length typing and DNA sequencing. Analysis of the ISR sequences showed that V. ichthyoenteri contains one types of polymorphic ISRs. The size of ISRs ranged 348bp length and not contains tRNA genes. Mutiple alignment of representative sequences from different Vibrio species revealed several domains of high sequence variability, and allowed to design species-specific primer for detection of Vibrio ichthyoenteri. PCR. The specific of the primer was examined using genomic DNA prepared from 19 different Vibrio species, isolated 18group Vibrio species. The results showed that the PCR reaction using species-specific primer designed in this study can be used to detect V. ichthyoenteri.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.10
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• pp.1550-1557
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• 2018

Objective: Circular RNAs (circRNAs) are a newfound class of non-coding RNA in animals and plants. Recent studies have revealed that circRNAs play important roles in cell proliferation, differentiation, autophagy and apoptosis during development. However, there are few reports about muscle development-related circRNAs in livestock. Methods: RNA sequencing analysis was employed to identify and annotate circRNAs from longissimus dorsi of sheep. Reverse transcription followed by real-time quantitative (q) polymerase chain reaction (PCR) analysis verified the presence of these circRNAs. Targetscan7.0 and miRanda were used to analyse the interaction of circRNA-microRNA (miRNA). To investigate the function of circRNAs, an experiment was conducted to perform enrichment analysis hosting genes of circRNAs using gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. Results: About 75.5 million sequences were obtained from RNA libraries of sheep skeletal muscle. These sequences were mapped to 729 genes in the sheep reference genome. We identified 886 circRNAs, including numerous circular intronic RNAs and exonic circRNAs. Reverse transcription PCR (RT-PCR) and DNA sequencing analysis confirmed the presence of several circRNAs. Real-Time RT-PCR analysis exhibited resistance of sheep circRNAs to RNase R digestion. We found that many circRNAs interacted with muscle-specific miRNAs involved in growth and development of muscle, especially circ776. The GO and KEGG enrichment analysis showed that hosting genes of circRNAs was involved in muscle cell development and signaling pathway. Conclusion: The study provides comprehensive expression profiles of circRNAs in sheep skeletal muscle. Our study offers a large number of circRNAs to facilitate a better understanding of their roles in muscle growth. Meanwhile, we suggested that circ776 could be analyzed in future study.

• Journal of Microbiology and Biotechnology
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• v.26 no.12
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• pp.2206-2213
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• 2016

Recently, several studies have revealed that commercial microbial identification systems do not accurately identify the uncommon causative species of candidiasis, including Candida famata, Meyerozyma guilliermondii, and C. auris. We investigated the accuracy of species-level identification in a collection of clinical isolates previously identified as C. famata (N = 38), C. lusitaniae (N = 1 2), and M. guilliermondii (N = 5) by the Vitek 2 system. All 55 isolates were re-analyzed by the Phoenix system (Becton Dickinson Diagnostics), two matrix-assisted laser desorption ionization-time of flight mass spectrometry analyzers (a Vitek MS and a Bruker Biotyper), and by sequencing of internal transcribed spacer (ITS) regions or 26S rRNA gene D1/D2 domains. Among 38 isolates previously identified as C. famata by the Vitek 2 system, the majority (27/38 isolates, 71.1%) were identified as C. tropicalis (20 isolates) or C. albicans (7 isolates) by ITS sequencing, and none was identified as C. famata. Among 20 isolates that were identified as C. tropicalis, 17 (85%) were isolated from urine. The two isolates that were identified as C. auris by ITS sequencing originated from ear discharge. The Phoenix system did not accurately identify C. lusitaniae, C. krusei, or C. auris. The correct identification rate for 55 isolates was 92.7% (51/55 isolates) for the Vitek MS and 94.6% (52/55 isolates) for the Bruker Biotyper, as compared with results from ITS sequencing. These results suggest that C. famata is very rare in Korea, and that the possibility of misidentification should be noted when an uncommon Candida species is identified.

• Reproductive and Developmental Biology
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• v.31 no.4
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• pp.227-233
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• 2007

This study was conducted to examine the mRNA expression of apoptosis-related and imprinted genes and methylation pattern of the differentially methylated region (DMR) of H19 gene in day 35 of SCNT pig fetuses. The day 35 of natural mating (control) or cloned (clone) pig fetuses were recovered from uterus. Endometrium from dam and liver from fetus were obtained, respectively. mRNA expression was evaluated by real-time PCR and methylation pattern was analyzed by bisulfite sequencing method. The Bcl-2 mRNA expression in clone was significantly lower than that of control (p<0.05). The mRNA expression of H19 gene in both endometrium and liver was significantly higher in clone than that of control, respectively (p<0.05). The level of IGF-2 mRNA in liver of clone was significantly lower than that of control (p<0.05), whereas the mRNA expression of IGF2-R gene in liver of clone was significantly higher than that of control (p<0.05). The DMR of H19 was lower methylation pattern in clone than that of control. These results suggest that the aberrant mRNA expression of apoptosis-related and imprinted genes and the lower DMR methylation pattern of imprinted gene may be closely related to the inadequate fetal development of cloned fetus.

• Microbiology and Biotechnology Letters
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• v.47 no.3
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• pp.401-411
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• 2019

All Idiomarina species are isolated from saline environments; microorganisms in such extreme habitats develop metabolic adaptations and can produce compounds such as proteases with an industrial potential. ARDRA and 16S rRNA gene sequencing are established methods for performing phylogenetic analysis and taxonomic identification. However, 16S-23S ITS is more variable than the 16S rRNA gene within a genus, and is therefore, used as a marker to achieve a more precise identification. In this study, ten protease producing Idiomarina strains isolated from the Peruvian salterns were characterized using biochemical and molecular methods to determine their bacterial diversity and industrial potential. In addition, comparison between the length and nucleotide sequences of a 16S-23S ITS region allowed us to assess the inter and intraspecies variability. Based on the 16S rRNA gene, two species of Idiomarina were identified (I. zobellii and I. fontislapidosi). However, biochemical tests revealed that there were differences between the strains of the same species. Moreover, it was found that the ITS contains two tRNA genes, $tRNA^{Ile(GAT)}$ and $tRNA^{Ala(TGC)}$, which are separated by an ISR of a variable size between strains of I. zobellii. In one strain of I. zobellii (PM21), we found nonconserved nucleotides that were previously not reported in the $tRNA^{Ala}$ gene sequences of Idiomarina spp. Thus, based on the biochemical and molecular characteristics, we can conclude that protease producing Idiomarina strains have industrial potential; only two I. zobellii strains (PM48 and PM72) exhibited the same properties. The differences between the other strains could be explained by the presence of subspecies.

• The Plant Pathology Journal
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• v.37 no.6
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• pp.619-631
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• 2021

Alfalfa mosaic virus (AMV), an economically important pathogen, is present worldwide with a very wide host range. This work reports for the first time the infection of Vinca minor and Wisteria sinensis with AMV using RNA sequencing and reverse transcription polymerase chain reaction confirmation. De novo assembly and annotating of contigs revealed that RNA1, RNA2, and RNA3 genomic fragments consist of 3,690, 2,636, and 2,057 nucleotides (nt) for IR-VM and 3,690, 2,594, and 2,057 nt for IR-WS. RNA1 and RNA3 segments of IR-VM and IR-WS closely resembled those of the Chinese isolate HZ, with 99.23-99.26% and 98.04-98.09% nt identity, respectively. Their RNA2 resembled that of Canadian isolate CaM and American isolate OH-2-2017, with 97.96-98.07% nt identity. The P2 gene revealed more nucleotide diversity compared with other genes. Genes in the AMV genome were under dominant negative selection during evolution, and the P1 and coat protein (CP) proteins were subject to the strongest and weakest purifying selection, respectively. In the population genetic analysis based on the CP gene sequences, all 107 AMV isolates fell into two main clades (A, B) and isolates of clade A were further divided into three groups with significant subpopulation differentiation. The results indicated moderate genetic variation within and no clear geographic or genetic structure between the studied populations, implying moderate gene flow can play an important role in differentiation and distribution of genetic diversity among populations. Several factors have shaped the genetic structure and diversity of AMV: selection, recombination/reassortment, gene flow, and random processes such as founder effects.