• Title/Summary/Keyword: Molecular sequence analyses

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Alternative splicing and expression analysis of High expression of osmotically responsive genes1 (HOS1) in Arabidopsis

  • Lee, Jeong-Hwan;Kim, Soo-Hyun;Kim, Jae-Joon;Ahn, Ji-Hoon
    • BMB Reports
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    • v.45 no.9
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    • pp.515-520
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    • 2012
  • High expression of osmotically responsive genes1 (HOS1), a key regulator of low temperature response and flowering time, encodes an E3 ubiquitin ligase in Arabidopsis. Here, we report characterization of a newly identified splice variant (HOS1-L) of HOS1. Comparative analyses revealed that HOS1-L has a longer 5' nucleotide sequence than that of the previously identified HOS1 (HOS1-S) and that its protein sequence was more conserved than that of HOS1-S in plants. HOS1-L transcripts were spatio-temporally more abundant than those of HOS1-S. The recovery rate of HOS1-S expression was faster than that of HOS1-L after cold treatment. Diurnal oscillation patterns of HOS1-L revealed that HOS1-L expression was affected by photoperiod. An in vitro pull-down assay revealed that the HOS1-L protein interacted with the ICE1 protein. HOS1-L overexpression caused delayed flowering in wild-type plants. Collectively, these results suggest regulation of HOS1 expression at the post-transcriptional level.

Molecular Identification of a Possible Causative Agent of Stomach Distension Syndrome in Rainbow Trout Onchorhynchus mykiss (무지개송어(Onchorhynchus mykiss) 위팽창증후군의 잠재적 원인체의 분자유전학적 동정)

  • Roh, Heyong Jin;Kim, Do-Hyung
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.50 no.5
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    • pp.624-629
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    • 2017
  • A rainbow trout Onchorhynchus mykiss farm located in Gangwon province, South Korea, experienced approximately 10% mortality in June 2017. Most diseased fish had a markedly distended, gas-filled stomach, and exhibited abnormal behavior at the water surface. In this study, we attempted to identify the cause of stomach distension syndrome in those rainbow trout. The stomach of most of the affected fish were full of unidentified gases and some exudate, and yeast was isolated from the stomach mucosa. Pure cultures of yeast were identified using a multilocus sequence typing scheme based on 18S rRNA, internal transcribed spacers, large subunit rRNA, and the gene encoding the largest subunit of RNA polymerase II (RPB1). The RPB1 gene sequences were compared with those of related species available in a database. The yeast was identified as Scheffersomyces coipomoensis (Candida coipomoensis) based on sequence analyses. This is the first study to reveal that Sch. coipomoensis is a potential causative agent of stomach distension syndrome in farmed rainbow trout. Our results will be helpful for future related studies, and indicate that farmers and stakeholders should observe this emerging disease closely.

Sensitive, Accurate PCR Assays for Detecting Harmful Dinoflagellate Cochlodinium polykrikoides Using a Specific Oligonucleotide Primer Set

  • Kim Chang-Hoon;Park Gi-Hong;Kim Keun-Yong
    • Fisheries and Aquatic Sciences
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    • v.7 no.3
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    • pp.122-129
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    • 2004
  • Harmful Cochlodinium polykrikoides is a notorious harmful algal bloom (HAB) species that is causing mass mortality of farmed fish along the Korean coast with increasing frequency. We analyzed the sequence of the large subunit (LSD) rDNA D1-D3 region of C. polykrikoides and conducted phylogenetic analyses using Bayesian inference of phylogeny and the maximum likelihood method. The molecular phylogeny showed that C. polykrikoides had the genetic relationship to Amphidinium and Gymnodinium species supported only by the relatively high posterior probabilities of Bayesian inference. Based on the LSU rDNA sequence data of diverse dinoflagellate taxa, we designed the C. polykrikoides-specific PCR primer set, CPOLY01 and CPOLY02 and developed PCR detection assays for its sensitive, accurate HAB monitoring. CPOLY01 and CPOLY02 specifically amplified C. polykrikoides and did not cross-react with any dinoflagellates tested in this study or environmental water samples. The effective annealing temperature $(T_{p})$ of CPOLY01 and CPOLY02 was $67^{\circ}C$. At this temperature, the conventional and nested PCR assays were sensitive over a wide range of C. polykrikoides cell numbers with detection limits of 0.05 and 0.0001 cells/reaction, respectively.

Isolation, Restriction Mapping, and Promoter Sequence Analysis of an Isoperoxidase Gene from Korean-Radish, Raphanus sativus L.

  • Park, Jong-Hoon;Kim, Soung-Soo
    • BMB Reports
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    • v.29 no.1
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    • pp.52-57
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    • 1996
  • A specific DNA fragment from Korean radish (Raphanus sativus L.) was amplified by performing PCR with oligonucleotide primers which correspond to the highly conserved regions of plant peroxidases. The size of the PCR product was ca. 400 bp, as expected from the known plant peroxidase genes. Comparison of the nucleotide and deduced amino acid sequences of the PCR product to those of other plant peroxidase-encoding genes revealed that the amplified fragment corresponded to the highly conserved region I and III of plant peroxidases. By screening a genomic library of Korean radish using the amplified fragment as a probe, two positive clones, named prxK1 and prxK2, were isolated. Restriction mapping studies indicated that the 5.2 kb Sail fragment of the prxK1 clone and the 4.0 kb EcoRI fragment of the prxK2 clone encode separate isoperoxidase genes. Analyses of the promoter region of the prxK1 clone shows that putative CAAT box, CMT box, and TGA1b binding sequence (5' TGACGT) are present 718 bp upstream from the start codon.

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Cloning and expression of trypsin-encoding cDNA from Blattella germanica and its possibility as an allergen

  • OCK Mee Sun;KIM Bong Jin;KIM Sun Mi;BYUN Kang Hyun
    • Parasites, Hosts and Diseases
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    • v.43 no.3 s.135
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    • pp.101-110
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    • 2005
  • In this study, the trypsin gene (bgtryp-1) from the German cockroach, Blattella germanica, was cloned via the immunoscreening of patients with allergies to cockroaches. Nucleotide sequence analysis predicted an 863 bp open reading frame which encodes for 257 amino acids. The deduced amino acid sequence exhibited $42-57\%$ homology with the serine protease from dust mites, and consisted of a conserved catalytic domain (GOSGGPLV). bgtryp-1 was determined by both Northern and Southern analysis to be a 0.9 kb, single-copy gene. SDS-PAGE and Western blotting analyses of the recombinant protein (Bgtryp-1) over-expressed in Escherichia coli revealed that the molecular mass of the expressed protein was 35 kDa, and the expressed protein was capable of reacting with the sera of cock-roach allergy patients. We also discussed the possibility that trypsin excreted by the digestive system of the German cockroach not only functions as an allergen, but also may perform a vital role in the activation of PAR-2.

Evaluation of taxonomic validity of four species of Acanthamoeba: A. divionensis, A. paradivionensis, A. mauritaniensis, and A. rhysodes, inferred from molecular analyses

  • LIU Hua;MOON Eun-Kyung;YU Hak-Sun;JEONG Hae-Jin;HONG Yeon-Chul;KONG Hyun-Hee;CHUNG Dong-Il
    • Parasites, Hosts and Diseases
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    • v.43 no.1 s.133
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    • pp.7-13
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    • 2005
  • The taxonomy of Acanthamoeba spp., an amphizoic amoeba which causes granulomatous amoebic encephalitis and chronic amoebic keratitis, has been revised many times. The taxonomic validity of some species has yet to be assessed. In this paper, we analyzed the morphological characteristics, nuclear 18s rDNA and mitochondrial 16s rDNA sequences and the Mt DNA RFLP of the type strains of four Acanthamoeba species, which had been previously designated as A. divionensis, A. parasidionensis, A. mauritaniensis, and A. rhysodes. The four isolates revealed characteristic group II morphology. They exhibited 18S rDNA sequence differences of $0.2-1.1\%$ with each other, but more than $2\%$ difference from the other compared reference strains. Four isolates formed a different clade from that of A. castellanii Castellani and the other strains in morphological group lion the phylogenetic tree. In light of these results, A. paradivionensis, A. divionensis, and A. mauritaniensis should be regarded as synonyms for A. rhysodes.

Genetic Diversity in the Coat Protein Genes of Prune dwarf virus Isolates from Sweet Cherry Growing in Turkey

  • Ozturk, Yusuf;Cevik, Bayram
    • The Plant Pathology Journal
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    • v.31 no.1
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    • pp.41-49
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    • 2015
  • Sweet cherry is an important fruit crop with increasing economical value in Turkey and the world. A number of viruses cause diseases and economical losses in sweet cherry. Prune dwarf virus (PDV), is one of the most common viruses of stone fruits including sweet cherry in the world. In this study, PDV was detected from 316 of 521 sweet cherry samples collected from 142 orchards in 10 districts of Isparta province of Turkey by double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA). The presence of PDV in ELISA positive samples was confirmed in 37 isolates by reverse transcription- polymerase chain reaction (RT-PCR) method. A genomic region of 862 bp containing the coat protein (CP) gene of PDV was re-amplified from 21 selected isolates by RT-PCR. Amplified DNA fragments of these isolates were purified and sequenced for molecular characterization and determining genetic diversity of PDV. Sequence comparisons showed 84-99% to 81-100% sequence identity at nucleotide and amino acid level, respectively, of the CP genes of PDV isolates from Isparta and other parts of the world. Phylogenetic analyses of the CP genes of PDV isolates from different geographical origins and diverse hosts revealed that PDV isolates formed different phylogenetic groups. While isolates were not grouped solely based on their geographical origins or hosts, some association between phylogenetic groups and geographical origins or hosts were observed.

Cytospora elaeagnicola sp. nov. Associated with Narrow-leaved Oleaster Canker Disease in China

  • Zhang, Linxuan;Alvarez, Lourdes V.;Bonthond, Guido;Tian, Chengming;Fan, Xinlei
    • Mycobiology
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    • v.47 no.3
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    • pp.319-328
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    • 2019
  • Cytospora is a genus including important phytopathogens causing severe dieback and canker diseases distributed worldwide with a wide host range. However, identification of Cytospora species is difficult since the currently available DNA sequence data are insufficient. Aside the limited availability of ex-type sequence data, most of the genetic work is only based on the ITS region DNA marker which lacks the resolution to delineate to the species level in Cytospora. In this study, three fresh strains were isolated from the symptomatic branches of Elaeagnus angustifolia in Xinjiang Uygur Autonomous Region, China. Morphological observation and multi-locus phylogenetic analyses (ITS, LSU, ACT and RPB2) support these specimens are best accommodated as a distinct novel species of Cytospora. Cytospora elaeagnicola sp. nov. is introduced, having discoid, nearly flat, pycnidial conidiomata with hyaline, allantoid conidia, and differs from its relatives genetically and by host association.

Morphological Identification and Phylogenetic Analysis of Laelapin Mite Species (Acari: Mesostigmata: Laelapidae) from China

  • Yang, Huijuan;Yang, Zhihua;Dong, Wenge
    • Parasites, Hosts and Diseases
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    • v.60 no.4
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    • pp.273-279
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    • 2022
  • Laelapinae mites are involved in transmission of microbial diseases between wildlife and humans, with an impact on public health. In this study, 5 mite members in the subfamily Laelapinae (laelapin mites; LM) were morphologically identified by light microscopy, and the phylogenetic relationship of LM was analyzed in combination with the sequence information of part of the LM cytochrome oxidase subunit I (cox1) gene. The morphological identification revealed that 5 mites belonged to the genera Laelaps and Haemolaelaps, respectively. Sequence analysis showed that the ratio of nonsynonymous mutation rate to synonymous mutation rate of LM was less than 1, indicating that the LM cox1 gene had undergone purifying selection. Phylogenetic analysis showed that the Laelapinae is a monophyletic group. The genera Haemolaelaps and Hyperlaelaps did not separated into distinct clades but clustered together with species of the genus Laelaps. Our morphological and molecular analyses to describe the phylogenetic relationships among different genera and species of Laelapinae provide a reference for the improvement and revision of the LM taxonomy system.

Molecular Detection of Phytoplasmas of the 16SrI and 16SrXXXII Groups in Elaeocarpus sylvestris Trees with Decline Disease in Jeju Island, South Korea

  • Geon-Woo, Lee;Sang-Sub, Han
    • The Plant Pathology Journal
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    • v.39 no.1
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    • pp.149-157
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    • 2023
  • Phytoplasmas were discovered in diseased Elaeocarpus sylvestris trees growing on Jeju Island that showed symptoms of yellowing and darkening in the leaves. Leaf samples from 14 symptomatic plants in Jeju-si and Seogwipo-si were collected and phytoplasma 16S rRNA was successfully amplified by nested polymerase chain reaction using universal primers. The sequence analysis detected two phytoplasmas, which showed 99.5% identity to 'Candidatus Phytoplasma asteris' and 'Ca. P. malaysianum' affiliated to 16SrI and 16SrXXXII groups, respectively. Through polymerase chain reaction-restriction fragment length polymorphism (RFLP) analyses using the AfaI (RsaI) restriction enzyme, the presence of two phytoplasmas strains as well as cases of mixed infection of these strains was detected. In a virtual RFLP analysis with 17 restriction enzymes, the 16S rRNA sequence of the 'Ca. P. asteris' strain was found to match the pattern of the 16SrI-B subgroup. In addition, the phytoplasmas in the mixed-infection cases could be distinguished using specific primer sets. In conclusion, this study confirmed mixed infection of two phytoplasmas in one E. sylvestris plant, and also the presence of two phytoplasmas (of the 16SrI and 16SrXXXII groups) in Jeju Island (Republic of Korea).