• Title/Summary/Keyword: clone identification

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Identification of a Tandemly Repented DNA Sequence Using Combined RAPD and FISH in Welsh Onion (Allium fistulosum)

  • Bong Bo Seo;Geum Sook Do;Seon Hee Lee
    • Animal cells and systems
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    • v.3 no.1
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    • pp.69-72
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    • 1999
  • A tandemly repeated DNA sequence was identified and characterized y the combined RAPD and FISH data from a total genomic DNA of Welsh onion (Allium fistulosum). A clone containing this repeating sequence was selected and sequenced. This repeating unit of 314 bp inserted into pAf 072 contained 54.1% adenine and thymine residues, and showed the primer sequence used, 5'-GAAACGGGTG-3', in both terminals of the sequence. Fluorescence in situ hybridization using this tandemly repeated sequence as a probe indicated that the detected sites were coincident with the major C-banded constitutive heterochromatin in the terminal regions of both arms of all 6 chromosomes.

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Studies on the Morphological, Anatomical and Physiological Characters in Populus alba × glandulosa and its Parent (Populus alba × glandulosa 와 그의 양친종(両親種)의 형태(形態), 해부학(解剖學), 생리학적(生理学的) 연구(硏究))

  • Kim, Chung Suk;Chon, Sang Keun;Hwang, Jin Sung
    • Journal of Korean Society of Forest Science
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    • v.49 no.1
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    • pp.11-31
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    • 1980
  • The morphological, anatomical and physiological traits were eximined for Populus alba ${\times}$ glandulosa which is an important planting species in Korea. The results obtained are as follows: 1. External characters in the leaf shape and chaff shape in the catkin were inherited as incomplete dominance but nectar gland was inherited as dominance. 2. Among the 15 selected clones, 9 clones were male, 2 clones female and 2 clones monoecious. 3. There were well-developed cork layers and bast fiber bundles in the bark. 4. Primordial leaves composed of 3 layers of cells and those undifferentiated into palisade and spongy parenchymas differed in its origin. 5. Leaf scare consisted of two kinds of tissues; one is connected to vascular bundle and the other not to vascular bundle. Tissues which had been connected to vascular bundle were isolated with only 2 or 3 layers of cork cells from the outside. 6. There was complicated arrangement in the vascular bundle of petioles. 7. Growth of the hybrid was sensitively influenced by external temperature, day-length and amount of light. In particular, it was apparent in height growth. 8. Flatness, loam soils and a $60{\times}60cm$ spacing might be best factors for the growth of P. alba ${\times}$ glandulosa. 9. The rooting of 15 clones was dependant upon external factors. 10. The growth of P. alba ${\times}$ glandulosa was best at around 80% of soil moisture content on the basis of plot water capacity. 11. Temperature difference between inside and outside stems below 100cm during the winter was the greatest at the south among seasons and among directions. 12. The sap movement was markedly influenced by air temperature, relative humidity in forest stand and moisture content in stem. 13. Total sugars in the cortex changed with season but did not differ in the dircetion of the stem. 14. Isoperoxidase variations in the leaf were different among 15 clones. Thus, it may be useful as a criterium for clonal identification. 15. The rate of soil moisture content decreased at a rapid slope was faster than that at a slow slope. Poor growth of P. alba ${\times}$ glandulosa at the slope was probably due to depletion of soil moisture.

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Identification of a Genetic Locus Related to Antivirus Production in Pseudomonas fluorescence strain Gpf01 Against Cucumber mosaic virus

  • Cho, Sae-Youll;Lee, Seon-Hwa;Park, Su-Jin;Choi, Kyu-Up;Cho, Jun-Mo;Hur, Jang-Hyun;Shrestha, Anupama;Lim, Chun-Keun
    • The Plant Pathology Journal
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    • v.25 no.1
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    • pp.77-85
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    • 2009
  • Pseudomonas fluorescens strain Gpf01, isolated from ginseng rhizosphere showed antiviral activity against Cucumber mosaic virus, when tested in a local host of CMV, Chenopodium amaranticolor. Transposon mutant library of Gpf01 was prepared using pGS9::Tn5 and the mutant Gpf01-RS19 was found to loose antiviral production. We developed primers from the flanking region of Tn5 and found a cosmid clone pAV1123, harboring 1.2 kb antiviral compound producing (avcf01) locus. When a sub-clone pPH9, which carried 9.3 kb region of pAV1123, was introduced into antivirus deficient P. fluorescens wild type strain B16, it exhibited antiviral activity. Using Tn3-gus mutagenesis and complementation analysis, it was found that the genes related to antiviral activity production resided in a 9.3 kb HindIII-HindIII fragment of pAV1123, indicating that the plasmid carries an essential genes promoting antiviral activity.

Development of Repetitive DNA Probes for Genetic Analysis of Phytophthora capsici (Phytophthora capsici의 유전적 특성 분석을 위한 Repetitive DNA Probe의 개발)

  • Song, Jeong-Young;Kim, Hong-Gi
    • The Korean Journal of Mycology
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    • v.30 no.1
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    • pp.66-72
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    • 2002
  • To develop DNA markers for analysis of genetic characteristics of Phytophthora capsici population, randomly selected clones from HindIII-digested genomic DNA library of P. capsici 95CY3119 were surveyed by hybridizing to Southern blots of HindIII-digested total genomic DNA of P. capsici. Probe DNAs inserted into selected individual clones strongly hybridized with HindIII digests of P. capsici. Among probes examined, PC9 revealed the repetitive and highly polymorphic bands to HindIII digests of inter-and intra-field P. capsici isolates. Genetic diversity of individual isolates was also clearly revealed in cluster analysis based on its band patterns. The other probe, PC22, was hybridized only to DNA from P. capsici and this was highly repetitive. However, there was no response to other Phytophthora species and Pythium sp. These DNA probes could be used as very useful markers in analysing genetic diversity and identification for P. capsici population throughout the world.

Identification of an Embryonic Growth Factor IGF-II from the Central Nervous System of the Teleost, Flounder, and Its Expressions in Adult Tissues

  • Kim, Dong-Soo;Kim, Young-Tae
    • Journal of Microbiology and Biotechnology
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    • v.9 no.1
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    • pp.113-118
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    • 1999
  • The insulin-like growth factor (IGF) is found in all vertebrates and its type-II molecule is regarded as a fundamental embryonic growth factor during development. We have firstly identified, in this study, a cDNA clone corresponding to IGF-II (flIGF-II) from the adult brain of the teleost, Paralichthys olivaceus. We also examined the tissue expression of flIGF-II in several adult tissues by RT-PCR. The flIGF-II cDNA contained a complete ORF consisting of 215 amino acids and one stop codon. Its molecular characteristics appear to be similar to the previously identified IGF-II molecules, in which a common primary structure exhibiting B, C, A, D, and E domains is evidently observed. This cDNA clone seems to be cleaved at $Ala_{52}$ for the $NH_2$-end signal peptide and appears to produce a 98 amino acid-long E-peptide from the $Arg^{118}$. The functional B-D domain regions, therefore, include 65 amino acids and is able to encode a 7.4-kDa protein. The most prominent structural difference between IGF-I and IGF-II was that the D domain of IGF-II exhibits a two-codon-deleted pattern compared to the 8 amino acid-containing IGF-I. The insulin family signature in the A domain and six cysteins forming three disulfide bridges between the B and A domains were evolutionary-conserved from teleosts to mammalian IGF-II. Interestingly, the E-peptide region appears to provide a distinct hallmark between teleosts in amino acid composition. The flIGF-II shows 85.1% of sequence identity to salmon and trout, 90.6% to tilapia, and 98.4% to perch in amino acid level. In tissue expressions of IGF-II, it is very likely that flIGF-II has a significant expression in the adult brain. However, liver seems to be the main source for IGF-II production, and relatively low signals were observed in the adult muscle and kidney. Taken together, it would be concluded that the functional region for IGF-II mRNA is highly similar in phylogeny and is evolutionary, conserved as a mediator for the growth of vertebrates.

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Genes of Rhodobacter sphaeroides 2.4.1 Regulated by Innate Quorum-Sensing Signal, 7,8-cis-N-(Tetradecenoyl) Homoserine Lactone

  • Hwang, Won;Lee, Ko-Eun;Lee, Jeong-Kug;Park, Byoung-Chul;Kim, Kun-Soo
    • Journal of Microbiology and Biotechnology
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    • v.18 no.2
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    • pp.219-227
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    • 2008
  • The free-living photoheterotrophic Gram-negative bacterium Rhodobacter sphaeroides possesses a quorum-sensing (QS) regulatory system mediated by CerR-CerI, a member of the LuxR-LuxI family. To identify the genes affected by the regulatory system, random lacZ fusions were generated in the genome of R. sphaeroides strain 2.4.1 using a promoter-trapping vector, pSG2. About 20,000 clones were screened and 23 showed a significantly different level of ${\beta}$-gal activities upon the addition of synthetic 7,8-cis-N-tetradecenoyl-homoserine lactone (RAI). Among these 23 clones, the clone showing the highest level of induction was selected for further study, where about a ten-fold increase of ${\beta}$-gal activity was exhibited in the presence of RAI and induction was shown to be required for cerR. In this clone, the lacZ reporter was inserted in a putative gene that exhibited a low homology with catD. A genetic analysis showed that the expression of the catD homolog was initiated from a promoter of another gene present upstream of the catD. This upstream gene showed a strong homology with luxR and hence was named qsrR (quorum-sensing regulation regulator). A comparison of the total protein expression profiles for the wild-type cells and qsrR-null mutant cells using two-dimensional gel electrophoresis and a MALDI-TOF analysis allowed the identification of sets of genes modulated by the luxR homolog.

Molecular Cloning and Identification of Novel Genes, Gomsin, Characteristically Expressed in Snailfish, Liparis tanakae (꼼치에서 특징적으로 발현되는 새로운 유전자 곰신의 분리 및 동정)

  • 송인선;이석근;손진기
    • Development and Reproduction
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    • v.6 no.1
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    • pp.7-16
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    • 2002
  • In order to obtain the specific genes of snailfish a subtracted cDNA library was constructed, and analysed by sequencing and GenBank search. Among them C90-171 clone was turned out to be genes showing low homology and nonredundant genes. This novel clone was named Gomsin(C90-171). Gomsin was shown to be intensely expressed in the epithelial cells, some mesenchymal cells, and sheaths of muscle bundles in the result of immunohistochemistry. In the cross reaction assay of Gomsin antibody against various human tissues, the Gomsin was strongly expressed in the ductal and acinar cells of salivary glands, which was similar to the expression patterns of proline-rich proteins(PRPs) of human. The antibody raised against the Gomsin was clearly cross-reacted with human salivary PRPs and also recombinant proteins of human PRPs in the Western blot and immunoprecipitation analysis. Contrast to the salivary PRPs, the Gomsin was not easily degraded in the mixed saliva, but rapidly attacked on the cultured keratocytes in vitro. The simulated protein structure of Gomsin was similar to the whorled pattern of PRPs, even though the amino acid sequence of Gomsin was quite different from those of PRPs. These data suggest that the Gomsin is a characteristic matrix protein in the skin and body of snailfish, which is also utilized for the tissue protection in the similar way to the PRPs of human muco-secretory organs.

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Identification of Genes for Biosynthesis of Antibacterial Compound from Pseudomonas fluorescens Bl6, and Its Activity Against Ralstonia solanacearum

  • Kim, Jin-Woo;Kim, Jung-Gun;Park, Byoung-Keun;Choi, Ok-Hee;Park, Chang-Seuk;Hwang, In-Gyu
    • Journal of Microbiology and Biotechnology
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    • v.13 no.2
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    • pp.292-300
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    • 2003
  • Pseudomonas fluorescens B16 is a plant glowth-prornoting rhizobacterium, which produces an antibacterial compound that is effective against plant root pathogens, such as Agrobacrerium tumefaciens and Raistonia solanacearum. We mutagenized the strain B16 with Omegon-Km and isolated six antibacterial-activity-deficient mutants. Two cosmid clones that hybridized with the mutant clones also were isolated from a genomic library of tile parent strain. Using deletion and complementation analyses, it was found that the biosynthesis genes resided in a 4.3-kb SalI-NarI fragment. When a plasmid clone carrying the fragment was introduced into P. fluorescens strain 1855.344, which does not exhibit any antibacterial activity, the transconjugants exhibited antibacterial activity, indicating that the plasmid clone carried all the genes essential for production of the antibacterial compound. DNA sequence analysis of the fragment identified four putative open reading frames (ORFs): orf1 through orf4 The deduced amino acid sequences of ORF1, ORF2, and ORF4 were similar to cystathionine gamma lyase, pyruvate formate-lyase activating enzyme, and transcriptional regulator, respectively, yet the amino acid sequence of ORF3 showed no similarities to any known proteins. It was also demonstrated that the antibacterial activity was responsible for biological control of the bacterial wilt caused by R. solanacearum.

Characterization of Homologous Defective Interfering RNA during Persistent Infection of Vero Cells with Japanese Encephalitis Virus

  • Yoon, Sung Wook;Lee, Sang-Yong;Won, Sung-Yong;Park, Sun-Hee;Park, Soo-Young;Jeong, Yong Seok
    • Molecules and Cells
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    • v.21 no.1
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    • pp.112-120
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    • 2006
  • It has been suggested that defective interfering (DI) RNA contributes to the persistence of Japanese encephalitis virus (JEV). In this study, we characterized molecular and biological aspects of the DI RNA and its relation to viral persistence. We identified a homologous DI virus intimately associated with JEV persistence in Vero cells. The production of DI RNA during undiluted serial passages of JEV coincided with the appearance of cells refractory to acute infection with JEV. We also established a Vero cell clone with a persistent JEV infection in which the DI RNA coreplicated efficiently at the expense of helper virus. The infectious virus yield of the clone fluctuated during its growth depending upon the amount of DI RNA accumulated in the previous replication cycle. Identification of the corresponding negative-sense RNA of the DI RNA indicated that the DI RNA functioned as a replication unit. Most of the DI RNA molecules retained their open reading frames despite a large deletion, encompassing most of the prM, the entire E, and the 5' half of the NS1 gene. Taken together, these observations suggest that the generation of homologous DI RNA during successive JEV acute infections in Vero cells probably participates actively in persistent JEV infection.

Isolation and Identification of a Histamine-degrading Barteria from Salted Mackerel (자반고등어에서 histamine 분해능을 가진 세균의 분리 동정)

  • Hwang Su-Jung;Kim Young-Man
    • Journal of Life Science
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    • v.15 no.5 s.72
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    • pp.743-748
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    • 2005
  • Histamine can be produced at early spoilage stage through decarboxylation of histidine in red-flesh fish by Proteus morganii, Hafnia alvei or Klebsiella pneumoniae. Allergic food poisoning is resulted from the histamine produced when the freshness of Mackerel degrades. Conversely it has been reported that there are bacteria which decompose histamine at the later stage. We isolated histamine decomposers from salted mackerel and studied the characteristics to help establish hygienic measure to prevent outbreak of salted mackerel food poisoning. All the samples were purchased through local supermarket. Histamine decomposers were isolated using restriction medium using histamine 10 species were selected. Identification of these isolates were carried out by the comparison of 16S rDNA partial sequence; as a result, we identified Pseudomonas putida strain RA2 and Halomonas marina, Uncultured Arctic sea ice bacterium clone ARKXV1/2-136, Halomonas venusta, Psychrobacter sp. HS5323, Pseudomonas putida KT2440, Rhodococcus erythropolis, Klebsiella terrigena (Raoultella terrigena), Alteromonadaceae bacterium T1, Shewanella massilia with homology of $100\%,{\;}100\%,{\;}99\%,{\;}99\%,{\;}99\%,{\;}99\%,{\;}100\%,{\;}95\%,{\;}99\%,{\;}and{\;}100\%$respectively. Turbidometry determination method and enzymic method were employed to determine the ability of histamine decomposition. Among those species Shewanella massilia showed the highest in ability of histamine decomposition. From these results we confirmed various histamine decomposer were present in salted mackerel product in the market.