• Journal of Aquaculture
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• v.7 no.1
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• pp.41-54
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• 1994

In order to evaluate the availability of lacZ as a reporter gene for producing transgenic mud loach, foreign DNA, bacterial \beta-galactosidase$ gene (lacZ) was microinjected into mud loach eggs and its insertion and expression were examined. X-gal based histochemical assay, fluorimetric analysis of \beta-galactosidase$ with 4-methylumbelliferyl-$\beta$-D-galactoside (MUG) and molecular biological examination using polymerase chain reaction (PCR), dot blot, southern blot and sequence analysis of PCR products were carried out to analyze both microinjected group and non-injected controls. The results are disccussed in this paper.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.20
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• pp.8883-8886
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• 2014

Background: Tetracycline is an antibiotic widely used for the treatment of Helicobacter pylori infection, but its effectiveness is decreasing due to increasing bacterial resistance. The aim of this study was to investigate the occurrence of 16S rRNA mutations associated with resistance or reduced susceptibility to tetracycline ofHelicobacter pylori by real-time PCR (RT-PCR) assays from culture. Materials and Methods: Tetracycline susceptibility and minimal inhibition concentration (MIC) was determined by the Epsilometer test (Etest) method. A LightCycler assay developed to detect these mutations was applied to DNA extracted from culture. The 16S rRNA of these isolates was sequenced and resistance-associated mutations were identified. From 104 isolates of H. pylori examined, 11 showed resistance to tetracycline. Results: LightCycler assay was applied to DNA extracted from 11 tetracycline-susceptible and 11 tetracycline resistance H. pylori isolates. In our study the sequencing of the H. pylori wild types in 16 s rRNA gene were AGA 926-928 with MIC (0.016 to $0.5{\mu}g/ml$), while the sequencing and MIC for resistant were GGA and AGC, (0.75 to $1.5{\mu}g/ml$), respectively. Also we found a novel mutation in 2 strains with $84^{\circ}C$ as their melting temperatures and exhibition of an A939C mutation. Conclusions: We conclude that real-time PCR is an excellent method for determination of H. pylori tetracycline resistance related mutations that could be used directly on biopsy specimens.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.5
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• pp.3001-3003
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• 2013

Objective: Spleen tyrosine kinase (Syk) is closely related to tumor invasion and metastasis, and has been shown to have potential inhibitory effects in tumors. In this study, we constructed a eukaryotic expression vector for Syk and analyzed its effects on invasive ability of the A549 non-small cell lung cancer cell line in vitro. Methods: A fragment of Syk was obtained by RT-PCR from human lung cancer cells and cloned into the expression vector pLNCXSyk. After restriction endonuclease digestion, PCR and DNA sequencing confirmation, the recombinant Syk expression plasmid was transfected into A549 human lung cancer cells using lipofectamine protocols. After selection, the cells stably expressed Syk. Detection of Syk expression of the cells by RT-PCR, and invasive ability were examined. Results: The eukaryotic expression plamid pLNCXSyk was constructed and expressed stably in the A549 human lung cancer cells. The RT-PCR results showed that Syk mRNA expression was upregulated significantly (P<0.05). Lower invasion through a basal membrane were apparent after transfection (P<0.05). Conclusions: A eukaryotic expression plasmid to cause Syk expression in lung cancer cells can obviously inhibit their invasive ability in vitro.

• IMMUNE NETWORK
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• v.8 no.3
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• pp.82-89
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• 2008

Background: Although a skin test is the primary option for detecting allergen-specific IgE in clinics, the serum IgE immunoassay is also important because it allows for the diagnosis of allergy without any accompanying adverse effect on the patient. However, the low detection limit of IgE levels by immunoassay may restrict the use of the method in some occasions, and improving its sensitivity would thus have a significant implication in allergy-immunology clinics. Methods: In this study, we attempted to detect specific serum IgE by using immuno-polymerase chain reaction (IPCR) which combines the antigen-antibody specificity of enzyme-linked immunosorbent assays (ELISAs) with the amplification power of PCR. Results: Our results demonstrated that Blo t5-specific serum IgE can be detected by IPCR with a 100-fold higher sensitivity than ELISA, and cross-reactivity of serum IgE to other mite allergens is able to be analyzed by using only $0.3{\mu}l$ of serum sample. Use of real-time IPCR seemed to permit more convenient determination of specific serum IgE as well. Conclusion: We believe that IPCR can serve as a valuable tool in determining specific serum IgE, especially when the amount of serum sample is limited.

• Korean Journal Plant Pathology
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• v.13 no.4
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• pp.239-243
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• 1997

In August 1996, phyllody disease of sesame (Sesamum indicum L.) caused by phytoplasmas was observed at Boeun, Chungbuk Province, Korea. Symptoms included extreme proliferation of growing tips and numerous small leaves, giving the infected plant a witche's-broom effect. Parts or all of the floral parts were transformed into green leaf-like structures, and little or no seeds were produced. Transmission Electron microscopy revealed the presence of phytoplasmas in the phloem sieve elements of infected plant. Since the infected sesame plants were growing near by phytoplasma infected jujubes (Zizyphus jujubu), we tried a polymerase chain reaction (PCR) technique to identify these two causal phytoplasmas. The DNA extracted from the stems of infected sesame plant was PCR-amplified using a primer set specific to 16S rRNA gene of known phytoplasmas. The amplification generated a 1.4kb band in both sesame samples and phytoplasma-infected jujubes, which also suggests the sesame plants were infected with phytoplasmas. The restriction digestion of the amplified band by four different enzymes, AluI, HaeIII, HinfI or TaqI revealed that the phytoplasmas infecting jujubes and sesame plants were of different groups.

• The Plant Pathology Journal
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• v.31 no.2
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• pp.123-131
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• 2015

Clavibacter michiganensis subsp. sepedonicus (Cms) multiplies very rapidly, passing through the vascular strands and into the stems and petioles of a diseased potato. Therefore, the rapid and specific detection of this pathogen is highly important for the effective control of the pathogen. Although several PCR assays have been developed for detection, they cannot afford specific detection of Cms. Therefore, in this study, a computational genome analysis was performed to compare the sequenced genomes of the C. michiganensis subspecies and to identify an appropriate gene for the development of a subspecies-specific PCR primer set (Cms89F/R). The specificity of the primer set based on the putative phage-related protein was evaluated using genomic DNA from seven isolates of Cms and 27 other reference strains. The Cms89F/R primer set was more specific and sensitive than the existing assays in detecting Cms in in vitro using Cms cells and its genomic DNA. This assay was also able to detect at least $1.47{\times}10^2copies/{\mu}l$ of cloned-amplified target DNA, 5 fg of DNA using genomic DNA or $10^{-6}$ dilution point of 0.12 at $OD_{600}$ units of cells per reaction using a calibrated cell suspension.

• The Plant Pathology Journal
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• v.16 no.3
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• pp.142-146
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• 2000

Ordered differential display using RT-PCR (ODD-PCR) was conducted to have a profile of the differently expressed genes between a hypovirulent strain of Cryphonectria parasitica (UEP1) and its isogenic wild type strain (EP155/2). ODD-PCR has advantages of high sensitivity, reproducibility, proportional representation, and limited number of primer combinations comparing with other differential display methods. RNAs were prepared from 1 and 5 day liquid culture of both hypovirulent and wild type strains, and were further evaluated with the marker genes of C. parasitica such as cryparin and mating factor MF2-1, which were already proven to be specifically down-regulated by the presence of mycovirus CHV1-713. ODD-PCR was conducted using those RNAs and expressed genes were categorized to five groups according to their temporal and quantitative expression patterns. Those fives groups are CPC, CPE, CPL, CPD, and CPU which represent constitutively-expressed, early-expressed, late-expressed, down-regulated, and up-regulated, respectively. Ninety two primer combinations out of a total of 192 have been tested so far. Among the twenty to fifty distinct bands per each reaction, an average of four to ten genes was identified as viral-regulated fungal genes. Those viral-specifc genes were further analyzed by DNA sequencing followed by homology search. Characterization of 30 clones including all five groups were conducted as a preliminary data and more are under investigation.

• Plant Resources
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• v.5 no.1
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• pp.59-69
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• 2002

Twenty-three plants of Japanese apricot (Prunus mume) were collected from several sites around Mountains JIRI in Korea. Japanese apricots having the different morphological features were evenly distributed in the groups made from the cluster analysis, indicating no geographic distributions but artificial vegetations in Korea. Japanese apricots were, as based on the PCR-RAPD techniques, clustered into the three groups; a group (prototype) having the five white petals with the five red sepals, a group (green type) having the five white petals with the five green sepals, and a group (hybrid type) having the more than five red petals with various colored sepals. The prototype apricots showed higher toxicities than other type apricot against bacteria and production of less compounds in TLC plates. The polypetal types of Japanes apricot were related to those of p. armebiaca in the characteristics of seed (the ruggedness), but also to be closed to those of p. armebiaca in PCR-RAPD analysis. The cluster analysis of the twenty three apricots and its related species calculated from the two primers were shown to distinguish relationships of cultivars within species, or of individual plants within cultivars, but also to display the two overlapping bands resulted from PCR-RAPD technique.

• Archives of Pharmacal Research
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• v.29 no.1
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• pp.88-95
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• 2006

Analysis of molecular nature of mitochondrial DNA (mtDNA) could be powerful marker for anthropological studies of modern populations. While population genetic studies on mtDNA have been reported for several ethnic groups, no such study has been documented for the Korean population. We surveyed mtDNA polymorphisms in the HVS I of noncoding D-loop region and its upstream region from 430 unrelated healthy Korean population by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and direct sequencing analysis. PCR product with 2,790 bp spanning the specific mtDNA region (mt13715-16504) was subjected to RFLP analysis using 6 restriction enzyme (Hinf I, Hae III, Alu I, Dde I, Mbo I, Rsa I). On the PAUP analysis of PCR-RFLP results, 38 mtDNA haplotypes (Hap 1-38) were detected in the Korean populations, which were classified into 11 haplogroups (Grp 1-11) of related haplotypes encompassing all 38 haplotypes. In comparison of sequencing data with Anderson's reference sequence, the transition type was more prevalent than the transversion type. Insertions or deletions were not found. In addition, three of the polymorphic sites (A16240C, A16351G, G16384A) in HVS-I region are determined newly. The polymorphic sites were distributed randomly in the region, though the frequency at each site was variable. Thus, this research might be required for the genealogical study of Orientals.

• Korean Journal of Agricultural Science
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• v.28 no.2
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• pp.92-98
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• 2001

Lactofenicin is an antibacterial peptide fragment (about 5 kD) derived from lactoferrin (80 kD) that displays the various biological functions. The production of a human lactoferricin (Lactoferricin H) in mouse HC11 mammary epithelial cells was achieved by placing its cDNA under the control of the bovine ${\beta}$-casein gene. To express lactoferricin H in this cell culture system, constructed a hybride-splice signal consisting of bovine ${\beta}$-casein intron I and rabbit ${\beta}$-globin intron II, and a DNA fragment spanning intron 8 of the bovine ${\beta}$-casein gene. Expression of lactofenicin H from this expression vector was identified by RT-PCR, northern and dot blot analysis. RT-PCR using total RNA of HC11 cells transfected with pBL1-cin expression vector yielded a product identified as having a size of the 150bp. Northern blot analysis was identified about 2.3 kb. In dot blot analysis, recombinant lactofenicin H was recognized with anti-human lactofrrnin polyclonal antibody.