• Title/Summary/Keyword: Molecular sequence analyses

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Identification of Marker Nucleotides for the Molecular Authentication of Araliae Continentalis Radix Based on the Analysis of Universal DNA Barcode, matK and rbcL, Sequences (범용성 DNA 바코드(matK, rbcL) 분석을 통한 독활(獨活) 유전자 감별용 Marker Nucleotide 발굴)

  • Kim, Wook Jin;Yang, Sungyu;Choi, Goya;Moon, Byeong Cheol
    • The Korea Journal of Herbology
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    • v.31 no.5
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    • pp.15-23
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    • 2016
  • Objectives : Araliae Continentalis Radix and Angelicae Pubescentis Radix have been used as the same medicinal name Korean and Chinese traditional medicines, respectively. The authentic Araliae Continentalis Radix is described only the root of Aralia continentalis in the Korean Pharmarcopoeia. However, the dried root of Angelica biserrata, Levisticum officinale, or Heracleum moellendorffii also has been distributed adulterants of Araliae Continentalis Radix. To develop a reliable method for identifying Araliae Continentalis Radix from adulterants, we carried out the analyses of universal DNA barcode sequences.Methods : Four plants species were collected from different habitate and nucleotide sequences of matK and rbcL were analyzed. The species-specific sequences and phylogenetic relationship were estimated using entire sequences of two DNA barcodes, respectively.Results : In comparative analysis of matK sequences, we were identified 104 positions of marker nucleotide for Ar. continentalis, 3 for An. biserrata, 4 for L. officinale and 8 for H. moellendorffii enough to distinguish individual species, respectively. Furthermore, we obtained marker nucleotides in rbcL at 42 positions for Ar. continentalis, 5 for An. biserrata and 2 for H. moellendorffii, but not for L. officinale. The phylogenetic tree of matK and rbcL were showed that all samples were clustered into four groups constituting homogeneous clades within the species.Conclusions : We confirmed that species-specific marker nucleotides of matK sequence provides distinct genetic information enough to identify four species. Therefore, we suggest that matK gene is useful DNA barcode for discriminating authentic Araliae Continentalis Radix from inauthentic adulterants.

Cloning of the non-virion (NV) of a Korean Isolate of Infectious Hematopoietic Necrosis and Identification of the Role of the NV in IHNV Replication (한국에서 분리된 전염성 조혈괴저 바이러스의 non-virion (NV) 단백질의 유전자 클로닝 및 바이러스 증식에서의 역할)

  • 문창훈;조화자;윤원준;박정재;박정민;김현주;도정완;이주양;임채렬
    • Korean Journal of Microbiology
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    • v.36 no.2
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    • pp.103-108
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    • 2000
  • We have cloned and analyzed cDNA coding for non-virion (NV) protein of the m V - P R T The NV gene contained 336 bp open readmg frame and encoded a protein of 11 1 amino acids with a molecular weight of 13.2 kDa. The deduced amino acid sequence of NV of IHNVPRT was found to be 90-95% identical to those of foreign isolates of IHNV. These results indicate that NV gene of the MNV is highly conserved among &ifferent strains of THNV Northern blot analyses revealed that the levels of NV gene expression were strongly elevated after 20 h post-infection. In order to identify the role of NV in the replication of MNV in fish cells, IHNVinfected cells were treated with antisense oligonucleotides. While IHNV-PRT exposed to glycoprotein (G) antisense oligonucleotide showed severely reduced growth, the growth of virus exposed to NV antisense oligonucleotide was not affected by NV antisense oligonucleotide, which suggests that NV is not essential for replication of IHNV in fish cells.

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The Heavy Metal Tolerant Soil Bacterium Achromobacter sp. AO22 Contains a Unique Copper Homeostasis Locus and Two mer Operons

  • Ng, Shee Ping;Palombo, Enzo A.;Bhave, Mrinal
    • Journal of Microbiology and Biotechnology
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    • v.22 no.6
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    • pp.742-753
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    • 2012
  • Copper-containing compounds are introduced into the environment through agricultural chemicals, mining, and metal industries and cause severe detrimental effects on ecosystems. Certain microorganisms exposed to these stressors exhibit molecular mechanisms to maintain intracellular copper homeostasis and avoid toxicity. We have previously reported that the soil bacterial isolate Achromobacter sp. AO22 is multi-heavy metal tolerant and exhibits a mer operon associated with a Tn21 type transposon. The present study reports that AO22 also hosts a unique cop locus encoding copper homeostasis determinants. The putative cop genes were amplified from the strain AO22 using degenerate primers based on reported cop and pco sequences, and a constructed 10,552 base pair contig (GenBank Accession No. GU929214). BLAST analyses of the sequence revealed a unique cop locus of 10 complete open reading frames, designated copSRABGOFCDK, with unusual separation of copCD from copAB. The promoter areas exhibit two putative cop boxes, and copRS appear to be transcribed divergently from other genes. The putative protein CopA may be a copper oxidase involved in export to the periplasm, CopB is likely extracytoplasmic, CopC may be periplasmic, CopD is cytoplasmic/inner membrane, CopF is a P-type ATPase, and CopG, CopO, and CopK are likely copper chaperones. CopA, B, C, and D exhibit several potential copper ligands and CopS and CopR exhibit features of two-component regulatory systems. Sequences flanking indicate the AO22 cop locus may be present within a genomic island. Achromobacter sp. strain AO22 is thus an ideal candidate for understanding copper homeostasis mechanisms and exploiting them for copper biosensor or biosorption systems.

Development of EST-SSR Markers for Evaluation of Genetic Diversity and Population Structure in Finger Millet (Eleusine coracana (L.) Gaertn.)

  • Lee, Myung Chul;Choi, Yu-Mi;Hyun, Do-Yoon;Lee, Sukyeung;Kim, Jin-Hee;Oh, Sejong
    • Proceedings of the Plant Resources Society of Korea Conference
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    • 2018.04a
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    • pp.105-105
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    • 2018
  • Finger millet, Eleusine coracana Gaertn., is more nutritious than other cereals and millets and widely cultivate in tropical regions of the world. However, status of its genetic diversity remained concealed due to lack of research work in this species. In recent years, microsatellites have become the most used markers for studying population genetic diversity. In present study, genetic diversity and structure of different populations of finger millet from Africa and South Asia was examined at molecular level using newly developed EST-Simple Sequence Repeat (EST-SSR) markers using a total of 1,927 ESTs of Eleusine coracana available in the NCBI database. In total, 46 primers produced 292 alleles in a size range of 100-500 bp and mean Polymorphism Information Content (PIC) and Marker Index (MI) were 0.372 and 1.04, respectively. 46 primers showed polymorphism and 21 primers were identified as having a PIC value above 0.5. Principal coordinates analysis and the dendrogram constructed out of combined data of both markers showed grouping of finger millet accessions to their respective area of collection. The 156 accessions was classified into four groups, such as three groups of Africa collection and one group of Asia. Results of present study can be useful in identifying diverse accessions and management of this plant resource. Moreover, the novel SSR markers developed can be utilized for various genetic analyses in this species in future.

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Efficiency to Discovery Transgenic Loci in GM Rice Using Next Generation Sequencing Whole Genome Re-sequencing

  • Park, Doori;Kim, Dongin;Jang, Green;Lim, Jongsung;Shin, Yun-Ji;Kim, Jina;Seo, Mi-Seong;Park, Su-Hyun;Kim, Ju-Kon;Kwon, Tae-Ho;Choi, Ik-Young
    • Genomics & Informatics
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    • v.13 no.3
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    • pp.81-85
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    • 2015
  • Molecular characterization technology in genetically modified organisms, in addition to how transgenic biotechnologies are developed now require full transparency to assess the risk to living modified and non-modified organisms. Next generation sequencing (NGS) methodology is suggested as an effective means in genome characterization and detection of transgenic insertion locations. In the present study, we applied NGS to insert transgenic loci, specifically the epidermal growth factor (EGF) in genetically modified rice cells. A total of 29.3 Gb (${\sim}72{\times}coverage$) was sequenced with a $2{\times}150bp$ paired end method by Illumina HiSeq2500, which was consecutively mapped to the rice genome and T-vector sequence. The compatible pairs of reads were successfully mapped to 10 loci on the rice chromosome and vector sequences were validated to the insertion location by polymerase chain reaction (PCR) amplification. The EGF transgenic site was confirmed only on chromosome 4 by PCR. Results of this study demonstrated the success of NGS data to characterize the rice genome. Bioinformatics analyses must be developed in association with NGS data to identify highly accurate transgenic sites.

Genetic Evolution and Characteristics of Ichthyotoxic Cochlodinium polykrikoides(Gymnodiniales, Dinophyceae) (어류치사성 Cochlodinium polykrikoides 적조생물의 유전적 진화 및 특성)

  • Cho, Eun-Seob;Jeong, Chang-Su
    • Journal of Life Science
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    • v.17 no.11
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    • pp.1453-1463
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    • 2007
  • This study presents a molecular phylogenetic analysis of the harmful dinoflagellate Cochlodinium polykrikoides, by use of partial sequence of small subunit (SSU) rRNA gene from most of the major taxa(24 species) in dinoflagellates. The class Dinophyceae clade formed a strong monophyletic relationship with C. polykrikoides and several taxa. On the basis of deeper nodes, the phylogenetic relationships placed C. polykrikoides closer to the order Prorocentrales rather than to the order Gymnodiniales, which was supported by a strong bootstrap value (100%) in the analyses of Neighbor-Joining and Parsimony methods. There is strong support for C. polykrikoides being placed in the same branch as Gymnodiniaceae and being connected in a clade with Prororcentrum micans among Prorocentrales. Morphological data show that C. polykrikoides is well associated with the genus Gyrodinium; however, this species is genetically closer to Gymnodinium than to Gyrodinium. The placement of C. polykrikoides always formed an independent branch separated from other dinoflagellates. In conclusion, planktonic P. micans plays an important role as an ancestor of Gymnodinium, whereas C. polykrikoides appears to be used an intermediate position between P. micans and Gymnodinium based on evolution.

Physicochemical and Microbiological Characterization of Protected Designation of Origin Ezine Cheese: Assessment of Non-starter Lactic Acid Bacterial Diversity with Antimicrobial Activity

  • Uymaz, Basar;Akcelik, Nefise;Yuksel, Zerrin
    • Food Science of Animal Resources
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    • v.39 no.5
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    • pp.804-819
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    • 2019
  • Ezine cheese is a non-starter and long-ripened cheese produced in the Mount of Ida region of Canakkale, Turkey, with a protected designation of origin status. Non-starter lactic acid bacteria (NSLAB) have a substantial effect on the quality and final sensorial characteristics of long-ripened cheeses. The dominance of NSLAB can be attributed to their high tolerance to the hostile environment in cheese during ripening relative to many other microbial groups and to its ability to inhibit undesired microorganisms. These qualities promote the microbiological stability of long-ripened cheeses. In this study, 144 samples were collected from three dairies during the ripening period of Ezine cheese. Physicochemical composition and NSLAB identification analyses were performed using both conventional and molecular methods. According to the results of a 16S rRNA gene sequence analysis, 13 different species belonging to seven genera were identified. Enterococcus faecium (38.42%) and E. faecalis (18.94%) were dominant species during the cheese manufacturing process, surviving 12 months of ripening together with Lactobacillus paracasei (13.68%) and Lb. plantarum (11.05%). The results indicate that NSLAB contributes to the microbiological stability of Ezine cheese over 12 months of ripening. The isolation of NSLAB with antimicrobial activity, potential bacteriocin producers, yielded defined collections of natural NSLAB isolates from Ezine cheese that can be used to generate specific starter cultures for the production of Ezine cheese (PDO).

Detection of Single Nucleotide Polymorphism in Human IL-4 Receptor by PCR Amplification of Specific Alleles

  • Hwang, Sue Yun;Kim, Seung Hoon;Hwang, Sung Hee;Cho, Chul Soo;Kim, Ho Youn
    • Animal cells and systems
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    • v.5 no.2
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    • pp.153-156
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    • 2001
  • A key aspect of genomic research in the “post-genome era”is to associate sequence variations with heritable phenotypes. The most common variations in the human genome are single nucleotide polymorphisms (SNPs) that occur approximately once in every 500 to 1,000 bases. Although analyzing the phenotypic outcome of these SNPs is crucial to facilitate large-scale association studies of genetic diseases, detection of SNPs from an extended number of human DNA samples is often difficult, labor-intensive and time-consuming. Recent development in SNP detection methods using DNA microarrays and mass spectrophotometry has allowed automated high throughput analyses, but such equipments are not accessible to many scientists. In this study, we demonstrate that a simple PCR-based method using primers with a mismatched base at the 3'-end provides a fast and easy tool to identify known SNPs from human genomic DNA in a regular molecular biology laboratory. Results from this PCR amplification of specific alleles (PASA) analysis efficiently and accurately typed the Q576R polymorphism of human IL4 receptor from the genomic DNAs of 29 Koreans, including 9 samples whose genotype could not be discerned by the conventiona1 PCR-SSCP (single strand conformation polymorphism) method. Given the increasing attention to disease-associated polymorphisms in genomic research, this alternative technique will be very useful to identify SNPs in large-scale population studies.

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Identification of Puccinia iridis on Iris domestica in Korea (범부채에서 녹병균 Puccinia iridis의 동정)

  • Choi, In-Young;Choi, Young-Joon;Kim, Jin-Young;Shin, Hyeon-Dong
    • The Korean Journal of Mycology
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    • v.47 no.1
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    • pp.89-94
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    • 2019
  • A rust fungus on Iris domestica (syn. Belamcanda chinensis) from China was previously identified as Puccinia iridis. Accordingly, the identity of the rust fungus on the same host species in Korea needs to be reexamned. Morphological characteristics of the Korean materials matched with P. iridis. Molecular phylogenetic analyses based on internal transcribed spacer and large subunit rDNA sequences of two representative materials confirmed the identification by high sequence similarities of 100% and 99% with the reference sequences available in NCBI GenBank database. Phylogenetic tree inferred from neighbor-joining method proved them to be clustered in P. iridis group. Thus, the rust fungus on I. domestica in Korea was determined as P. iridis. On the other hand, a previous record that Puccinia belamcandae is another rust agent on I. domestica in Korea should be reconfirmed in future.

Taxonomy of Arthrinium minutisporum sp. nov., Pezicula neosporulosa, and Acrocalymma pterocarpi: New Records from Soil in Korea

  • Das, Kallol;Lee, Seung-Yeol;Choi, Hyo-Won;Eom, Ahn-Heum;Cho, Young-Je;Jung, Hee-Young
    • Mycobiology
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    • v.48 no.6
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    • pp.450-463
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    • 2020
  • The strains 17E-042, 17E-039, and NC13-171 belong to Ascomycota and were isolated from soil collected from Sancheong-gun and Yeongam-gun, Korea. The strain 17E-042 produced white mycelial colonies that developed a sienna color with a round margin on potato dextrose agar (PDA), and the reverse side developed a light sienna color. Morphologically, this strain was similar to the strains of Arthrinium phragmites and A. hydei, but the shorter conidial size of the newly identified strain (17E-042) was distinct. The strain 17E-039 produced macroconidia that were pale yellow to orange-brown, elongated-ellipsoid to oblong, round at both ends, primarily straight but sometimes slightly curved, 0-septate, thin-walled, and filled with numerous droplets, having diameters of 20.4-34.3 × 8.0-12.0 ㎛. And the strain NC13-171 formed hyaline to light brown chlamydospores, solitary or in a chain. Multigene phylogenetic analyses were conducted using sequence data obtained from internal transcribed spacer (ITS) regions, 28S rDNA large subunit (LSU), β-tubulin (TUB2), translation elongation factor 1-alpha (TEF1-α), and RNA polymerase II large subunit (RPB2) genes. The results of molecular phylogeny, the detailed descriptions and illustrations of each species strongly support our proposal that these strains from soil in Korea be designated as Arthrinium minutisporum sp. nov. and two new records of Pezicula neosporulosa and Acrocalymma pterocarpi.