• Title/Summary/Keyword: Fragment size

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Genetic Variation of Rice Populations Estimated Using nrDNA ITS Region Sequence

  • Wang, Dong;Hong, Soon-Kwan
    • Korean Journal of Plant Resources
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    • v.27 no.3
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    • pp.249-255
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    • 2014
  • The rice belonging to Oryza sativa is not only has significant economic importance, for it is the major source of nutrition for about 3 billion all around the world. But also plays a vital role as a model organism, because it has a number of advantages to be a model plant, such as efficient transformation system and small genome size. Many methods and techniques have been conducted to attempt to distinguish different Oryza sativa species, such as amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR) and so on. However, studies using sequence analysis of internal transcribed spacer (ITS), a region of ribosomal RNA has not been reported until now. This study was undertaken with an aim to understand the phylogenetic relationships among sixteen isolates of Oryza sativa collected from abroad and fifteen isolates collected from Korea, using ribosomal RNA (rRNA) internal transcribed spacer (ITS) sequences to compare the phylogeny relationships among different Oryza sativa species. The size variation obtained among sequenced nuclear ribosomal DNA (nrDNA) ITS region ranged from 515bp to 1000bp. The highest interspecific genetic distance (GD) was found between Sfejare 45 (FR12) and Anapuruna (FR15). Taebong isolate showed the least dissimilarity of the ITS region sequence with other thirty isolates. This consequence will help us further understanding molecular diversification in intra-species population and their phylogenetic analysis.

Design and Expression of High Nutritional Peptide (HEAAE) in E. coli

  • Kim, Jae-Ho;Lee, Chang-Kook;Hong, Bum-Shik
    • Journal of Microbiology and Biotechnology
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    • v.7 no.2
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    • pp.132-137
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    • 1997
  • A novel protein (HEAAE, High Essential Amino Acid Encoding Protein), rich in essential amino acids ($75{\%}$ of total), was designed and constructed in our laboratory. The designed peptides were analyzed by SYBLE and stable secondary and tertiary structures were predicted. The monomeric form (HEAAE-1) of the protein consists of 20 amino acid residues with four additional amino acids comprising a potential ${\beta}$-turn (HEAAE-4). Size exclusion analysis demonstrated that the monomer is self-aggregates in aqueous solution to form higher ordered multimeric structures, which are very reminiscent of natural plant storage proteins. The DNA encoding this amino acid sequence was synthesized, and from this monomeric gene fragment (heaae-1), the stable tetrameric form of the gene (heaae-4) was generated by subcloning into the E. coli expression vector pKK223-3. A clear 6 kDa polypeptide band corresponding to the molecular weight of the dimeric form (HEAAE-2) was detected. The smeared band which appeared around the molecular weight corresponding to HEAAE-4 of 11 kDa suggested that the tetramer form of this protein might be processed into smaller size products.

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Field Studios of In-situ Aerobic Cometabolism of Chlorinated Aliphatic Hydrocarbons

  • Semprini, Lewts
    • Proceedings of the Korean Society of Soil and Groundwater Environment Conference
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    • 2004.04a
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    • pp.3-4
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    • 2004
  • Results will be presented from two field studies that evaluated the in-situ treatment of chlorinated aliphatic hydrocarbons (CAHs) using aerobic cometabolism. In the first study, a cometabolic air sparging (CAS) demonstration was conducted at McClellan Air Force Base (AFB), California, to treat chlorinated aliphatic hydrocarbons (CAHs) in groundwater using propane as the cometabolic substrate. A propane-biostimulated zone was sparged with a propane/air mixture and a control zone was sparged with air alone. Propane-utilizers were effectively stimulated in the saturated zone with repeated intermediate sparging of propane and air. Propane delivery, however, was not uniform, with propane mainly observed in down-gradient observation wells. Trichloroethene (TCE), cis-1, 2-dichloroethene (c-DCE), and dissolved oxygen (DO) concentration levels decreased in proportion with propane usage, with c-DCE decreasing more rapidly than TCE. The more rapid removal of c-DCE indicated biotransformation and not just physical removal by stripping. Propane utilization rates and rates of CAH removal slowed after three to four months of repeated propane additions, which coincided with tile depletion of nitrogen (as nitrate). Ammonia was then added to the propane/air mixture as a nitrogen source. After a six-month period between propane additions, rapid propane-utilization was observed. Nitrate was present due to groundwater flow into the treatment zone and/or by the oxidation of tile previously injected ammonia. In the propane-stimulated zone, c-DCE concentrations decreased below tile detection limit (1 $\mu$g/L), and TCE concentrations ranged from less than 5 $\mu$g/L to 30 $\mu$g/L, representing removals of 90 to 97%. In the air sparged control zone, TCE was removed at only two monitoring locations nearest the sparge-well, to concentrations of 15 $\mu$g/L and 60 $\mu$g/L. The responses indicate that stripping as well as biological treatment were responsible for the removal of contaminants in the biostimulated zone, with biostimulation enhancing removals to lower contaminant levels. As part of that study bacterial population shifts that occurred in the groundwater during CAS and air sparging control were evaluated by length heterogeneity polymerase chain reaction (LH-PCR) fragment analysis. The results showed that an organism(5) that had a fragment size of 385 base pairs (385 bp) was positively correlated with propane removal rates. The 385 bp fragment consisted of up to 83% of the total fragments in the analysis when propane removal rates peaked. A 16S rRNA clone library made from the bacteria sampled in propane sparged groundwater included clones of a TM7 division bacterium that had a 385bp LH-PCR fragment; no other bacterial species with this fragment size were detected. Both propane removal rates and the 385bp LH-PCR fragment decreased as nitrate levels in the groundwater decreased. In the second study the potential for bioaugmentation of a butane culture was evaluated in a series of field tests conducted at the Moffett Field Air Station in California. A butane-utilizing mixed culture that was effective in transforming 1, 1-dichloroethene (1, 1-DCE), 1, 1, 1-trichloroethane (1, 1, 1-TCA), and 1, 1-dichloroethane (1, 1-DCA) was added to the saturated zone at the test site. This mixture of contaminants was evaluated since they are often present as together as the result of 1, 1, 1-TCA contamination and the abiotic and biotic transformation of 1, 1, 1-TCA to 1, 1-DCE and 1, 1-DCA. Model simulations were performed prior to the initiation of the field study. The simulations were performed with a transport code that included processes for in-situ cometabolism, including microbial growth and decay, substrate and oxygen utilization, and the cometabolism of dual contaminants (1, 1-DCE and 1, 1, 1-TCA). Based on the results of detailed kinetic studies with the culture, cometabolic transformation kinetics were incorporated that butane mixed-inhibition on 1, 1-DCE and 1, 1, 1-TCA transformation, and competitive inhibition of 1, 1-DCE and 1, 1, 1-TCA on butane utilization. A transformation capacity term was also included in the model formation that results in cell loss due to contaminant transformation. Parameters for the model simulations were determined independently in kinetic studies with the butane-utilizing culture and through batch microcosm tests with groundwater and aquifer solids from the field test zone with the butane-utilizing culture added. In microcosm tests, the model simulated well the repetitive utilization of butane and cometabolism of 1.1, 1-TCA and 1, 1-DCE, as well as the transformation of 1, 1-DCE as it was repeatedly transformed at increased aqueous concentrations. Model simulations were then performed under the transport conditions of the field test to explore the effects of the bioaugmentation dose and the response of the system to tile biostimulation with alternating pulses of dissolved butane and oxygen in the presence of 1, 1-DCE (50 $\mu$g/L) and 1, 1, 1-TCA (250 $\mu$g/L). A uniform aquifer bioaugmentation dose of 0.5 mg/L of cells resulted in complete utilization of the butane 2-meters downgradient of the injection well within 200-hrs of bioaugmentation and butane addition. 1, 1-DCE was much more rapidly transformed than 1, 1, 1-TCA, and efficient 1, 1, 1-TCA removal occurred only after 1, 1-DCE and butane were decreased in concentration. The simulations demonstrated the strong inhibition of both 1, 1-DCE and butane on 1, 1, 1-TCA transformation, and the more rapid 1, 1-DCE transformation kinetics. Results of tile field demonstration indicated that bioaugmentation was successfully implemented; however it was difficult to maintain effective treatment for long periods of time (50 days or more). The demonstration showed that the bioaugmented experimental leg effectively transformed 1, 1-DCE and 1, 1-DCA, and was somewhat effective in transforming 1, 1, 1-TCA. The indigenous experimental leg treated in the same way as the bioaugmented leg was much less effective in treating the contaminant mixture. The best operating performance was achieved in the bioaugmented leg with about over 90%, 80%, 60 % removal for 1, 1-DCE, 1, 1-DCA, and 1, 1, 1-TCA, respectively. Molecular methods were used to track and enumerate the bioaugmented culture in the test zone. Real Time PCR analysis was used to on enumerate the bioaugmented culture. The results show higher numbers of the bioaugmented microorganisms were present in the treatment zone groundwater when the contaminants were being effective transformed. A decrease in these numbers was associated with a reduction in treatment performance. The results of the field tests indicated that although bioaugmentation can be successfully implemented, competition for the growth substrate (butane) by the indigenous microorganisms likely lead to the decrease in long-term performance.

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Modified T-RFLP Methods for Taxonomic Interpretation of T-RF

  • Lee, Hyun-Kyung;Kim, Hye-Ryoung;Mengoni, Alessio;Lee, Dong-Hun
    • Journal of Microbiology and Biotechnology
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    • v.18 no.4
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    • pp.624-630
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    • 2008
  • Terminal restriction fragment length polymorphism (T-RFLP) is a method that has been frequently used to survey the microbial diversity of environmental samples and to monitor changes in microbial communities. T-RFLP is a highly sensitive and reproducible procedure that combines a PCR with a labeled primer, restriction digestion of the amplified DNA, and separation of the terminal restriction fragment (T-RF). The reliable identification of T-RF requires the information of nucleotide sequences as well as the size of T-RF. However, it is difficult to obtain the information of nucleotide sequences because the T-RFs are fragmented and lack a priming site of 3'-end for efficient cloning and sequence analysis. Here, we improved on the T-RFLP method in order to analyze the nucleotide sequences of the distinct T-RFs. The first method is to selectively amplify the portion of T-RF ligated with specific oligonucleotide adapters. In the second method, the termini of T-RFs were tailed with deoxynucleotides using terminal deoxynucleotidyl transferase (TdT) and amplified by a second round of PCR. The major T-RFs generated from reference strains and from T-RFLP profiles of activated sludge samples were efficiently isolated and identified by using two modified T-RFLP methods. These methods are less time consuming and labor-intensive when compared with other methods. The T-RFLP method using TdT has the advantages of being a simple process and having no limit of restriction enzymes. Our results suggest that these methods could be useful tools for the taxonomic interpretation of T-RFs.

Cloning of the Endoglucanase Gene from Actinomyces sp. 40 in Escherichia coli and Some Properties of the Gene Products

  • Min, Hae-Ki;Choi, Yun-Jaie;Cho, Kwang-Keun;Ha, Jong-Kyu;Woo, Jung-Hee
    • Journal of Microbiology and Biotechnology
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    • v.4 no.2
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    • pp.102-107
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    • 1994
  • The $\beta$-1,4-endoglucanase gene from Actinomyces sp. 40 was cloned into Escherichia coli DH5$\alpha$ with pUC19. Chromosomal DNA from Actinomyces sp. 40 was cleaved with the restriction enzyme Sau3AI and ligated into pUC19 for the transformation of Escherichia coli DH5$\alpha$. Positive clones of $\beta$-1,4-endoglucanase gene were detected as the clear zones on a medium supplemented with carboxymethylcellulose (CMC). This transformant possessed a single plasmid, designated pDS1, which contained the vector DNA and a 3.5 kilobase (kb) Sau3AI insertion fragment encoding endoglucanase. The size of the cloned fragment was reduced to 2.0 kb. The endoglucanase activity produced by the E. coli DH5$\alpha$ (pDS6) was higher than that of Actinomyces sp. 40 strain. The optimum pH and temperature of the cloned enzyme were pH 4.0$\sim$5.0 and 55$^{\circ}C$, respectively. The cloned enzyme was stable at 55$^{\circ}C$ or below and in buffer ranging from pH 4.0 to 7.0. The enzyme degraded CMC but did not degrade xylan, cellobiose, and methyl-umbelliferylcellobiopyranoside (MUC).

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Technical Development for Large DNA Fragment Transformation in Plants

  • Park, Su-Ryun;Seo, Mi-Suk;Lee, Sang-Kug;Park, Jee-Young;Kim, Hye-Ran;Lee, Hyo-Yeon;Bang, Jae-Wook;Lim, Yong-Pyo
    • Journal of Plant Biotechnology
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    • v.2 no.2
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    • pp.89-96
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    • 2000
  • For large DNA fragment transformation in dicots and monocots, BIBAC2 vector system was applied to Arabidopsis thaliana and Oryza sativa L. cv. Jinmi as a model plant, respectively. For Arabidopsis, the Th1 gene in T23L3 BAC clone whose size is about 90 kb was used as the target gene source for transformation. Because T23L3 BAC clone was originally constructed in pBelloBAC11, the target gene was reconstructed into BIBAC2. As the results of reconstruction, 476 colonies were survived in selection medium containing 40 mg/L kanamycin. In colony hybridization analysis, 24 out of 476 colonies exhibited positive signals. In the pulsed-field gel electrophoresis analysis, 11 out of 24 positive clones exhibited the band at the location of 90 kb. In Southern hybridization, positive signal band at the location of 90 kb was observed in all 11 transformants. Using these verified clones, Agrobacterium-mediated transformation was applied to Arabidopsis thaliana th1-201 mutant for genetic complementation test. Twelve thousands T$_1$ seeds were harvested, and antibiotic selection test is being analyzed to verify whether these seeds were transformed. for rice, COR356 that contains 150 kb human genomic DNA in a BIBAC2 vector was used as the target gene. As the results of transformation, 151 out of 210 co-cultivated calli were survived in selection medium containing 5 mg/L hygromycin, and 45 out of 151 survived calli were regenerated into plants. Transformation efficiency was 21.6%. Progeny test using 71 seeds is being analyzed now. These results provide the potential that large DNA fragments can be transferred into both dicots and monocot by Agrobacterium-mediate d transformation system.

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Cloning of Genes for the Biosynthesis of Glutathione from E. coIi K-12 (E.coli K-12 균주로부터 글루타치온 합성 유전자의 클로닝)

  • 남용석;박영인;이세영
    • Microbiology and Biotechnology Letters
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    • v.19 no.6
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    • pp.575-582
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    • 1991
  • To increase the production of glutathione by the expression of recombinant gsh plasmids, two genes responsible for the biosynthesis of glutathione were isolated and cloned. To clone a gshI gene, the GS903 mutant strain, which is deficient in $\gamma$-glutamylcysteine synthetase activity, has been raised. A gshI gene was cloned using pBR322 plasmid as a 3.6 Kb PstI DNA fragment isolated from E. coli K-12 chromosomal DNA. Also a gshIl gene was cloned using pUC13 plasmid as a 2.2 Kb PstI-BamHI DNA fragment. To study the effects of plasmid copy number and passenger DNA size on the expression levels of the gsh genes, various recombinant plasmids containing different sets of genes were constructed. The expression levels of the gsh genes were increased approximately twice higher in pUC series plasmids than that in pBR322 plasmid. But the sizes of the passenger DNA containing the gsh genes in the vector plasmid did not affect on the expression levels of the gsh genes.

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Generation and Expression in Plants of a Single-Chain Variable Fragment Antibody Against the Immunodominant Membrane Protein of Candidatus Phytoplasma Aurantifolia

  • Shahryari, F.;Safarnejad, M.R.;Shams-Bakhsh, M.;Schillberg, S.;Nolke, G.
    • Journal of Microbiology and Biotechnology
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    • v.23 no.8
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    • pp.1047-1054
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    • 2013
  • Witches' broom of lime is a disease caused by Candidatus Phytoplasma aurantifolia, which represents the most significant global threat to the production of lime trees (Citrus aurantifolia). Conventional disease management strategies have shown little success, and new approaches based on genetic engineering need to be considered. The expression of recombinant antibodies and fragments thereof in plant cells is a powerful approach that can be used to suppress plant pathogens. We have developed a single-chain variable fragment antibody (scFvIMP6) against the immunodominant membrane protein (IMP) of witches' broom phytoplasma and expressed it in different plant cell compartments. We isolated scFvIMP6 from a naïve scFv phage display library and expressed it in bacteria to demonstrate its binding activity against both recombinant IMP and intact phytoplasma cells. The expression of scFvIMP6 in plants was evaluated by transferring the scFvIMP6 cDNA to plant expression vectors featuring constitutive or phloem specific promoters in cassettes with or without secretion signals, therefore causing the protein to accumulate either in the cytosol or apoplast. All constructs were transiently expressed in Nicotiana benthamiana by agroinfiltration, and antibodies of the anticipated size were detected by immunoblotting. Plant-derived scFvIMP6 was purified by affinity chromatography, and specific binding to recombinant IMP was demonstrated by enzyme-linked immunosorbent assay. Our results indicate that scFvIMP6 binds with high activity and can be used for the detection of Ca. Phytoplasma aurantifolia and is also a suitable candidate for stable expression in lime trees to suppress witches' broom of lime.

Construction of Recombinant DNA for Purification of the Gag-Pro Transframe Protein of Human T-cell Leukemia Virus Type I (HTLV-I) (Human T-cell Leukemia Virus Type I (HTLV-I) 의 Gag-Pro Transframe 단백질 정제를 위한 재조합 DNA 의 제작)

  • 남석현
    • Korean Journal of Microbiology
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    • v.30 no.6
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    • pp.466-471
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    • 1992
  • To determine the site at which -1 ribosomal frameshifting occurs within the gag-pro overlap of HTL V-I. DNA fragment corresponding to a portion of the gene overlap was cloned into a SP6 vector. The resultant plasmid harbors the hybrid gene consisting of a synthetic gene encoding 5 amino acids derived from chick prelysozyme including the initiator methionine plus 141 nucleotides of gag-pro overlapping region followed by Staphylococcus aurcus protein A gene fragment. In vitro transcription by SP6 RNA polymerase with this DNA template made an abundant amount of single species mRNA. Cell-free translation programmed with the RNA transcribed in vitro yielded a polypeptide of 21 kDal in size. which could be purified into homogeneity by IgG-Sepharose affinity chromatography. In vitro system described in this study must be useful for rapid purification and sequencing of the Gag-Pro transframe protein. allowing to determine the exact frameshift site on mRNA and to identify the tRNA involved in frameshifting event for the expression of pro gene.

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Preliminary Analysis of Molecular Biological Methods for Stock Identification of Small Yellow Croaker(Pseudosciaena polyactis) in the Yellow Sea (황해산 참조기(Pseudosciaena polyactis)의 계군 분석을 위한 분자생물학적 방법 검정)

  • HUE Hoi-Kwon;HWANG Gyu-Lin;LEE Yong-Chul;CHANG Chung-Soon
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.25 no.6
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    • pp.474-484
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    • 1992
  • The stock identification of small yellow croaker. Pseudosciaena Polyactis from Mokpo area was carried out using molecular biological methods such as mt-DNA restriction fragment length polymorphism(RFLP) and the N-terminal fragment polymorphism of muscle actin obtained after protease digestion. The entire mt-DNA genomic size from the small yellow croaker at Mokpo area was estimated to be about $16\pm0.2$ Kb. Furthermore, fourteen restriction endonucleases revealed a total of 37 restriction sites to the mt-DNA molecule, however, eight of the fourteen enzymes showed a significant restriction site variation. Six of the enzymes examined produced a single restriction profile for all individuals surveyed, indicating that they don't react on the same mt-DNA obtained from small yellow croaker. The Staphylococcus aureus $V_8$ protease is able to cleave the muscle actin of small yellow croaker and to yield a N-terminal peptide of 26 and 16 KDa, respectively.

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