• Environmental Engineering Research
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• v.14 no.1
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• pp.63-67
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• 2009

Escherichia coli K-12 (E. coli K-12) is a representative indicator globally used for distinguishing and monitoring dynamic fates of pathogenic microorganisms in the environment. This study investigated how to most critically quantify lacZ ($\beta$-galactosidase) gene in E. coli K-12 by two different real-time polymerase chain reaction (real-time PCR) in association with three different DNA extraction practices. Three DNA extractions, i.e., sodium dodecyl sulfate (SDS)/proteinase K, magnetic beads and guanidium thiocyanate (GTC)/silica matrix were each compared for extracting total genomic DNA from E. coli K-12. Among them, GTC/silica matrix and magnetic beads beating similarly worked out to have the highest (22-23 ng/${\mu}L$) concentration of DNA extracted, but employing SDS/proteinase K had the lowest (10 ng/${\mu}L$) concentration of DNA retrieved. There were no significant differences in the quantification of the copy numbers of lacZ gene between SYBR Green I qPCR and QProbe-qPCR. However, SYBR Green I qPCR obtained somewhat higher copy number as $1{\times}10^8$ copies. It was decided that GTC/silica matrix extraction or magnetic beads beating in combination with SYBR Green I qPCR can be preferably applied for more effectively quantifying specific gene from a pure culture of microorganism.

• BMB Reports
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• v.29 no.4
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• pp.308-313
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• 1996

An 1.4 kb of the fad3 cDNA encoding microsomal linoleic acid desaturase catalyzing the conversion of linoleic acid (18:2, ${\omega}-6$) to linolenic acid (18:2, ${\omega}-3$) was introduced into tobacco plants by the Agrobacterium-mediated plant transformation, Among the transgenic tobacco plants conferring kanamycin resistance, five transformants showing increment in unsaturated fatty acid contents were selected and further analyzed for the transgenecity, In genomic Southern blot analyses, copy numbers of the integrated fad3 DNA in chromosomal DNA of the five transgenic tobacco plants were varied among the transgenic lines. By Northern blot analyses, the abundancy of the fad3 mRNA transcript directed by Cauliflower Mosaic Virus 35S promoter was consistent with the relative copy number of the fad3 DNA integrated in the chromosome of transgenic tobacco plants. When compared with the wild type, accumulation of linolenic acid in transgenic tobacco roots was elevated 3.7- to 4.7-fold showing a corresponding decrease in the linoleic acid contents; however, slight increments for linolenic acid were noticed in transgenic leaf tissues. These results indicated that the elevated level of fad3 expression is achieved in transgenic tobacco plants.

• Horticultural Science & Technology
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• v.32 no.3
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• pp.375-381
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• 2014

Transgenic potato was generated to express recombinant 5 repeated ${\beta}$-amyloid ($A{\beta}$) peptides, potential antigens to be applied as a preventive accine for Alzheimer's disease using Agrobacterium mediated transformation. The $A{\beta}$ peptides were fused to the human IgG Fc fragment enhancing protein and KDEL, which is the endoplasmic reticulum (ER) retention signal ($5A{\beta}$-FcK). The $5A{\beta}$-FcK, was expressed under the control of the duplicated 35S promoter. PCR analysis confirmed the presence of the transgene in several transgenic potato lines. Southern blot analysis showed only a single gene copy number in transgenic line 22, whereas multiple gene copy numbers were shown for transgenic lines 31 and 44. Northern blot analysis showed that line 22 had stronger mRNA levels when compared to lines 31 and 44. Immunoblot analysis confirmed that the $5A{\beta}$-FcK protein was expressed in the transgenic potato plant. These results indicate that $5A{\beta}$ fused to Fc can be expressed in potato plants.

• Journal of Plant Biology
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• v.39 no.1
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• pp.49-55
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• 1996

We have studied the DNA of Allium sativum L. with respect to highly repetitive sequences. Fast reassociated DNA fragments expected to be highly repetitive sequences based on $C_{o}t$ curve were isolated and characterized. Their copy numbers were approximately $10^{5}~10^{7}$ per haploid genome. Nucleotide sequences analysis of six candidates reveals that their G/C content were low, 25-40% and typical patterns of repeating sequences exist. Repeat sequences were used as probes to access restriction fragment length polymorphism (RFLP) of genomic DNAs of four local clones, Tanyang, Mungyong, So san, and Uisong. The hybridization pattern were very similar among these four local clones.clones.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.3
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• pp.196-200
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• 2004

The supra molecular weight poly(〔R〕-3-hydroxybutyrate) (PH B), having a molecular weight greater than 2 million Da, has recently been found to possess improved mechanical properties compared with the normal molecular weight PHB, which has a molecular weight of less than 1 million Da. However, applications for this PHB have been hampered due to the difficulty of its production. Reported here, is the development of a new metabolically engineered Escherichia coli strain and its fermentation for high level production of supra molecular weight PHB. Recombinant E. coli strains, harboring plasm ids of different copy numbers containing the Alcaligenes latus PHB biosynthesis genes, were cultured and the molecular weights of the accumulated PHB were compared. When the recombinant E. coli XL1-Blue, harboring a medium-copy-number pJC2 containing the A. latus PHB biosynthesis genes, was cultivated by fed-batch culture at pH 6.0, supra molecular weight PHB could be produced at up to 89.8 g/L with a productivity of 2.07 g PHB/L-h. The molecular weight of PHB obtained under these conditions was as high as 22 MDa, exceeding by an order of magnitude the molecular weight of PHB typically produced in Ralstonia eutropha or recombinant E. coli.

• Microbiology and Biotechnology Letters
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• v.40 no.1
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• pp.76-81
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• 2012

Removal of methane, benzene and toluene was evaluated in a lab-scale biocover packed with a soil mixture of forest soil and earthworm cast (75:25 weight ratio). The bacterial community in the biocover was characterized using quantitative real-time PCR and terminal restriction fragment length polymorphism. Methane was removed at the upper layer of the biocover (-0.1 ~ -0.4 m), where the oxygen concentration was remarkably lower. The average removal efficiencies for methane and benzene/toluene were 90% and 99%, respectively. The pmoA gene copy numbers, responsible for methane oxidation, in the upper layer were higher than those in the lower layer. While type I methanotrohs dominated the lower layer, type II methanotrophs, such as Methylocystis and Methylosinus, were noted to be predominant in the upper layer. Benzene and toluene were removed from the lower layer (-0.6 ~ -0.9 m) as well as the upper layer. Moreover, the tmoA gene copy number, responsible for benzene/toluene oxidation, seen in the upper layer was not significantly different from those seen in the lower layer. These results suggest that a biocover packed with a soil and earthworm cast mixture is a promising method which could be utilized for the control of methane and volatile organic compounds such as benzene and toluene.

• Korean Journal of Agricultural Science
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• v.49 no.4
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• pp.795-807
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• 2022

The development and commercialization of industrial genetically modified (GM) organisms is actively progressing worldwide, highlighting an increased need for improved safety management protocols. We sought to establish an environmental monitoring method, using real-time polymerase chain reaction (PCR) and propidium monoazide (PMA) treatment to develop a quantitative detection protocol for living GM microorganisms. We developed a duplex TaqMan quantitative PCR (qPCR) assay to simultaneously detect the selectable antibiotic gene, ampicillin (AmpR), and the single-copy Escherichia coli taxon-specific gene, D-1-deoxyxylulose 5-phosphate synthase (dxs), using a direct cell suspension culture. We identified viable engineered E. coli cells by performing qPCR on PMA-treated cells. The theoretical cell density (true copy numbers) calculated from mean quantification cycle (Cq) values of PMA-qPCR showed a bias of 7.71% from the colony-forming unit (CFU), which was within ±25% of the acceptance criteria of the European Network of GMO Laboratories (ENGL). PMA-qPCR to detect AmpR and dxs was highly sensitive and was able to detect target genes from a 10,000-fold (10^-4) diluted cell suspension, with a limit of detection at 95% confidence (LOD_95%) of 134 viable E. coli cells. Compared to DNA-based qPCR methods, the cell suspension direct PMA-qPCR analysis provides reliable results and is a quick and accurate method to monitor living GM E. coli cells that can potentially be released into the environment.

• Genomics & Informatics
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• v.10 no.2
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• pp.81-87
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• 2012

Large-scale copy number variants (CNVs) in the human provide the raw material for delineating population differences, as natural selection may have affected at least some of the CNVs thus far discovered. Although the examination of relatively large numbers of specific ethnic groups has recently started in regard to inter-ethnic group differences in CNVs, identifying and understanding particular instances of natural selection have not been performed. The traditional $F_{ST}$ measure, obtained from differences in allele frequencies between populations, has been used to identify CNVs loci subject to geographically varying selection. Here, we review advances and the application of multinomial-Dirichlet likelihood methods of inference for identifying genome regions that have been subject to natural selection with the $F_{ST}$ estimates. The contents of presentation are not new; however, this review clarifies how the application of the methods to CNV data, which remains largely unexplored, is possible. A hierarchical Bayesian method, which is implemented via Markov Chain Monte Carlo, estimates locus-specific $F_{ST}$ and can identify outlying CNVs loci with large values of FST. By applying this Bayesian method to the publicly available CNV data, we identified the CNV loci that show signals of natural selection, which may elucidate the genetic basis of human disease and diversity.

• Journal of Life Science
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• v.20 no.2
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• pp.176-182
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• 2010

Magnaporthe oryzae is destructive plant-pathogenic fungus and causes rice blast. The pathogen uses several mechanisms to circumvent the inhibitory actions of fungicides. ATP-binding cassette (ABC) transporters are known to provide protection against toxic compounds in the environment. PC facilitated bioinformatic analysis, particularly with respect to accessing and extracting database information and domain identification. We predicted ABC transporter genes from the M. oryzae genome sequence with computation and bioinformatics tools. A total of thirty three genes were predicted to encode ABC transporters. Three of thirty three putative genes corresponded to three known ABC transporter genes (ABC1, ABC2 and ABC3). Copy numbers of the ABC transporter genes were proven by Southern blot analysis, which revealed that twenty genes tested exist as a single copy. We amplified the DNA complementary to RNA corresponding to eleven of these by reverse transcriptase polymerase chain reaction.

• Clinical and Experimental Pediatrics
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• v.57 no.3
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• pp.117-124
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• 2014

Purpose: Administration of antiretroviral drugs to mothers and infants significantly decreases mother-to-child human immunodeficiency virus (HIV) transmission; cesarean sections and discouraging breastfeeding further decreases this risk. The present study confirmed the HIV status of babies born to mothers infected with HIV and describes the characteristics of babies and mothers who received preventive treatment. Methods: This study retrospectively analyzed medical records of nine infants and their mothers positive for HIV who gave birth at Korea University Ansan Hospital, between June 1, 2003, and May 31, 2013. Maternal parameters, including HIV diagnosis date, CD4+ count, and HIV ribonucleic acid (RNA) copy number, were analyzed. Infant growth and development, HIV RNA copy number, and HIV antigen/antibody test results were analyzed. Results: Eight HIV-positive mothers delivered nine babies; all the infants received antiretroviral therapy. Three (37.5%) and five mothers (62.5%) were administered single- and multidrug therapy, respectively. Intravenous zidovudine was administered to four infants (50%) at birth. Breastfeeding was discouraged for all the infants. All the infants were negative for HIV, although two were lost to follow-up. Third trimester maternal viral copy numbers were less than 1,000 copies/mL with a median CD4+ count of $325{\mu}L$ ($92-729{\mu}L$). Among the nine infants, two were preterm (22.2%) and three had low birth weights (33.3%). Conclusion: This study concludes that prophylactic antiretroviral therapy, scheduled cesarean section, and prohibition of breastfeeding considerably decrease mother-to-child HIV transmission. Because the number of infants infected via mother-to-child transmission may be increasing, studies in additional regions using more variables are necessary.