• Food Science of Animal Resources
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• v.25 no.2
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• pp.203-209
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• 2005

Real time-polymerase chain reaction (RT-PCR) is currently considered as the most sensitive method to detect low abundant DNAs in samples. Compared to conventional PCR, real-time PCR has a high reliability because of excluding false-positive results and can allow a simultaneous faster detection and quantification of target DNAs. This study was carried out to identify the Hanwoo (Korean native cattle) beef by genotyping after DNA extraction of commercial beef in 41 restaurants. Since Hanwoo, Holstein and imported cattle meat have different patterns in the MC1R gene associated with the coat colors of cattles (C-type, C/T-type or T-type), we could identify the genotype using real-time PCR The result of real-time PCR assay for beef samples in 41 restaurants which are asserted to sell Hanwoo beef only, showed that 29 of 41 samples were Hanwoo beef gene type (T-type) and 12 of 41 samples were Holstein or imported cattle gene type (C-type or C/T-type). Therefore, the proportion of Han-woo beef was $70.7\%$ and the proportion of Holstein or imported cattle meat was $29.3\%(C/T-type; 12.2\%,\;C-type; 17.1\%)$.

• Toxicological Research
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• v.32 no.1
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• pp.81-87
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• 2016

Aflatoxin B1 (AFB1) is produced by Aspergillus flavus growing in feedstuffs. Early detection of maize contamination by aflatoxigenic fungi is advantageous since aflatoxins exert adverse health effects. In this study, we report the development of an optimized conventional PCR for AFB1 detection and a rapid, sensitive and simple screening Real-time PCR (qPCR) with SYBR Green and two pairs of primers targeting the aflR genes which involved aflatoxin biosynthesis. AFB1 contaminated maize samples were divided into three groups by the toxin concentration. Genomic DNA was extracted from those samples. The target genes for A. flavus were tested by conventional PCR and the PCR products were analyzed by electrophoresis. A conventional PCR was carried out as nested PCR to verify the gene amplicon sizes. PCR-RFLP patterns, obtained with Hinc II and Pvu II enzyme analysis showed the differences to distinguish aflatoxin-producing fungi. However, they are not quantitative and need a separation of the products on gel and their visualization under UV light. On the other hand, qPCR facilitates the monitoring of the reaction as it progresses. It does not require post-PCR handling, which reduces the risk of cross-contamination and handling errors. It results in a much faster throughout. We found that the optimal primer annealing temperature was $65^{\circ}C$. The optimized template and primer concentration were $1.5{\mu}L\;(50ng/{\mu}L)$ and $3{\mu}L\;(10{\mu}M/{\mu}L)$ respectively. SYBR Green qPCR of four genes demonstrated amplification curves and melting peaks for tub1, afIM, afIR, and afID genes are at $88.0^{\circ}C$, $87.5^{\circ}C$, $83.5^{\circ}C$, and $89.5^{\circ}C$ respectively. Consequently, it was found that the four primers had elevated annealing temperatures, nevertheless it is desirable since it enhances the DNA binding specificity of the dye. New qPCR protocol could be employed for the determination of aflatoxin content in feedstuff samples.

• Journal of Life Science
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• v.25 no.2
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• pp.249-255
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• 2015

Schisandrae fructus [Schizandra chinensis (Turcz.) Baillon] is a medicinal herb widely used for treating various inflammatory and immune diseases in East Asian countries. The Schisandrae Semen essential oil (SSeo) from this plant has pharmacological activities, including antioxidant, antimicrobial, and antitumoral activities. Nevertheless, the biological activities and underlying molecular mechanisms of the potential anti-cancer effects of this oil remain unclear. In the present study, we investigated the potential inhibition of apoptosis signaling pathways by SSeo in human leukemia U937 cells and evaluated the underlying molecular mechanism. Exposure to SSeo resulted in a concentration-dependent growth inhibition due to apoptosis, which was verified by DNA fragmentation, the presence of apoptotic bodies, and an increase in the sub-G1 ratio. Induction of apoptotic cell death by SSeo was correlated with the down-regulation of members of the inhibitor of apoptosis protein (IAP) family (including X-linked inhibitor of apoptosis protein (XIAP), cIAP-1, and surviving) and anti-apoptotic Bcl-2, and with up-regulation of death receptor (DR) 4 and DR5, depending on dosage. SSeo treatment also induced Bid truncation, mitochondrial dysfunction, proteolytic activation of caspase-3, -8 and -9, and concomitant degradation of activated caspase-3 target proteins such as poly (ADP-ribose) polymerase. Taken together, these findings suggest that SSeo may be a potential chemotherapeutic agent for use in the control of human leukemia cells. Further studies are needed to identify its active compounds.

• Journal of Life Science
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• v.26 no.10
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• pp.1182-1188
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• 2016

Membrane-associated guanylate kinase inverted-3 (MAGI-3) is a member of the family of membrane-associated guanylate kinases (MAGUKs). MAGI-3 modulates the kinase activity of protein kinase B (PKB)/AKT through interactions with phosphatase and tensin homolog (PTEN)/MMAC. Coxsackievirus B3 (CVB3) is a common causative agent of acute myocarditis and chronic dilated cardiomyopathy. Activation of AKT and extracellular signal-regulated kinases 1/2 (ERK1/2) is essential for CVB3 replication, but the relation between MAGI-3 signaling and CVB3 replication is not well understood. This study investigated the role of MAGI-3 in CVB3 infection and replication. MAGI-3 was overexpressed in HeLa cells by polyethylenimine (PEI) transfection. To optimize the transfection conditions, different ratios of plasmid DNA to PEI concentrations were used. MAGI-3 and empty plasmid DNA were transfected into the HeLa cells. MAGI-3 overexpression alone was not sufficient to efficiently activate AKT. However, expression of the CVB3 capsid protein VP1 dramatically increased in the HeLa cells overexpressing MAGI-3 24 h after CVB3 infection. In addition, the activities of AKT and ERK were significantly induced in the CVB3-infected MAGI-3 cells overexpressing HeLa. These results demonstrate that MAGI-3 expression upregulates CVB3 replication through AKT and ERK signaling activation. MAGI-3 may be an important target to control CVB3 replication.

• Korean Journal of Microbiology
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• v.36 no.4
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• pp.254-258
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• 2000

Invasion process of bacterial cell into intestinal epithelium is important in Salmonella infection. The invasion is induced by the proteins secreted by type III secretion appratus of Salmonella. It has been known that the proteins do not have N-terminal signal peptide existing in general secreted proteins. Recent studies on Yersinia reported that secretion signal of type III appratus may lie on 5'end secondary structure of mRNA of secreted protein. In this study, we constructed translational fusion of ompR and sopE, encoding type III secretion protein of Salmonella, and observed secretion of the fusion protein for investigating the secretion signal of Salmonella type III appratus. The sopE DNA fragments of the translational fusion contain the region of promoter and from start code to tenth or to fifth code. These translational fusions indicate that type III secretion signal of Salmonella is located on 5'end of mRNA encoding secreted protein. We constructed prototype of secretion vector using this signal to produce useful foreign protein.

• Korean Journal of Microbiology
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• v.44 no.4
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• pp.277-281
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• 2008

HIV affects many organ systems. Patients with HIV infection have substantially increased risk of developing various cancers, primarily by opportunistic infection with oncogenic viruses due to their immunocompromised status. However, extensive evidence also indicates that the viral protein, Tat itself, may playas a major factor in the development of AIDS-related neoplasms. The molecular mechanism underlying Tat's oncogenic activity may include deregulation of cellular genes. Therefore, in this study, we examined the effect of HIV-l Tat on CD99 as one of the target cellular genes, which is a well-known tumor marker in several cancers. By using established HeLa clones that are stably expressing Tat, we found that CD99 is upregulated by endogenous Tat, whereas STAT3 is down regulated. Upon the screening of genes differentially expressed between Tat-stable cells and the control cells by using the gene fishing technique, DEG, we detected 3 genes which expression is affected by the presence of Tat. Furthermore, the methylation specific PCR analysis of the stably Tat expressing cell lines revealed that the CD99 promoter is de methylated in the presence of Tat. Taken together, these results open a potential role of CD99 in AIDS-related oncogenesis via epigenetic regulation by HIV-1 Tat.

• Development and Reproduction
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• v.10 no.3
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• pp.177-184
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• 2006

Retinol-binding protein(RBP) plays an important role in the specific transport of retinol to target cells through the blood stream in higher vertebrates. In order to clarify the effects of 4-nonylphenol(4-NP) on RBP mRNA expression in the rockfish, Sebastes schlegeli which is common in coastal waters of Korea and commercially important species, the cDNA library was constructed from the liver, and a partial fragment of the RBP gene was cloned. The deduced amino acid sequence from the RBP mRNA showed a high homology to the amino acid sequence from Sparus aurata(80%), Oncorhynchus mykiss(72%) or Anguilla anguilla(78%). Effects of 4-NP on RBP and vitellogenin(VTG) mRNA expression level in rockfish were examined by the northern blot analysis. In female and male rockfish injected with 4-NP(10 mg/kg BW, lower dose), there was no changes in the levels of VTG mRNA expression in the liver. The RBP mRNA levels, however, decreased at 48 hours after the injection in male. In the rockfish injected with 4-NP(25 mg/kg BW, higher dose), the level of VTG mRNA expression increased after 24 hours, regardless of sex. The level of RBP mRNA expression decreased at 48 hours after the injection in both sexes. These data indicate that estrogenic mimics such as 4-NP exhibit a contrasting effect on RBP and VTG gene expression in rockfish.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.12
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• pp.1801-1808
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• 2019

Objective: As an iconic symbol of Qinghai-Tibetan Plateau and of high altitude, yak are subjected to hypoxic conditions that challenge aerobic metabolism. Matrix metalloproteinases-3 (MMP3) is assumed to be a key target gene of hypoxia-inducible factor-$1{\alpha}$ that function as a master regulator of the cellular response to hypoxia. Therefore, the aim of this investigation was to identify the DNA polymorphism of MMP3 gene in domestic yak and to explore its possible association with high-altitude adaptation. Methods: The single-nucleotide polymorphisms (SNPs) genotyping and mutations scanning at the MMP3 locus were conducted in total of 344 individuals from four domestic Chinese yak breeds resident at different altitudes on the Qinghai-Tibetan Plateau, using high-resolution melting analysis and DNA sequencing techniques. Results: The novel of SNPs rs2381 $A{\rightarrow}G$ and rs4331 $C{\rightarrow}G$ were identified in intron V and intron VII of MMP3, respectively. Frequencies of the GG genotype and the G allele of SNP rs2381 $A{\rightarrow}G$ observed in high-altitude Pali yak were significantly higher than that of the other yak breeds resident at middle or low altitude (p<0.01). No significant difference was mapped for SNP rs4331 $C{\rightarrow}G$ in the yak population (p>0.05). Haplotype GC was the dominant among the 4 yak breeds, and Pearson correlation analysis showed that the frequencies of GC was significantly lower in Ganan (GN), Datong (DT), and Tianzhu white yaks (TZ) compared with Pali (PL) yak. The two SNPs were in moderate linkage disequilibrium in high-altitude yaks (PL) but not in middle-altitude (GN, DT) and low-altitude (TZ) yaks. Conclusion: These results indicate that MMP3 may have been subjected to positive selection in yak, especially that the SNP rs2381 $A{\rightarrow}G$ mutation and GC haplotypes might contribute to adaptation for yak in high-altitude environments.

• Journal of Life Science
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• v.29 no.9
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• pp.1047-1054
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• 2019

Transposable elements (TEs) occupy approximately 45% of the human genome and can enter functional genes randomly. During evolutionary radiation, multiple copies of TEs are produced by duplication events. Those elements contribute to biodiversity and phylogenomics. Most of them are controlled by epigenetic regulation, such as methylation or acetylation. Every species contains their own specific mobile elements, and they are divided into DNA transposons and retrotransposons. Retrotransposons can be divided by the presence of a long terminal repeat (LTR). They show various biological functions, such as promoter, enhancer, exonization, rearrangement, and alternative splicing. Also, they are strongly implicated to genomic instability, causing various diseases. Therefore, they could be used as biomarkers for the diagnosis and prognosis of diseases such as cancers. Recently, it was found that TEs could produce miRNAs, which play roles in gene inhibition through mRNA cleavage or translational repression, binding seed regions of target genes. Studies of TE-derived miRNAs offer a potential for the expression of functional genes. Comparative analyses of different types of miRNAs in various species and tissues could be of interest in the fields of evolution and phylogeny. Those events allow us to understand the importance of TEs in relation to biological roles and various diseases.

• Research in Plant Disease
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• v.26 no.4
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• pp.195-201
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• 2020

Rice bacterial leaf blight (BLB) by Xanthomonas oryzae pv. oryzae (Xoo) is considered to be one of the major rice diseases steadily occurring around the rice-producing countries. In this study, we developed a recombinase polymerase amplification (RPA) assay for the rapid, convenient and specific diagnosis of Xoo by targeting Xoo-specific transposase A gene. As the target gene can be amplified in 10 min without DNA extraction process and special equipment for temperature control, RPA for BLB can be useful and practical component for on-site diagnosis.