• Food Science of Animal Resources
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• v.40 no.4
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• pp.628-635
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• 2020

The objective of this study was to detect three non-halal meat products consisted of dog, pork, and rat species in meatball using novel multiplex-PCR with 12S rRNA gene as target sites. A total of 33 self-made meatballs were used, and they were grouped into eleven types of meatball based on meat species origin contained in the meatballs. Each type consisted of three meatballs. Extraction of genomic DNA from the meatballs was used as a DNA template for simplex-, duplex-, and multiplex-PCR processes. The result of simplex-PCR, duplex-PCR, and multiplex-PCR showed that the 12S rRNA primer gene successfully amplified DNA for each species bovine, dog, pig, and rat, which are respectively indicated by 155, 244, 357, and 491 bp of DNA bands. In addition, multiplex-PCR with 12S rRNA gene primers can be uniquely and accurately used for detection bovine, dog, pig, and rat species on beef meatball in one reaction.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.6
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• pp.763-768
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• 2006

Genetic and pharmacological studies in mice have demonstrated a complementary role for the melanocortin 4 receptor (MC4R) in the control of food intake, energy balance and body weight. This study was designed to investigate the associations of a MC4R gene polymorphism on chicken growth and body composition traits in broiler lines divergently selected for abdominal fat. A SNP (G54C) was found in CDS region of chicken MC4R gene. The analysis of the least squares and variance revealed a significant association between the G54C SNP and BW, CW and SL at 7 wk of age, and there were significant differences in different genotypes (p<0.05). The results from protein secondary structure prediction and tertiary structure prediction showed that it appeared a helix in $13^{th}$ amino acid and two strands at $14^{th}$ and $15^{th}$ amino acid in mutant protein, respectively. It maybe induce the change of the activity or function of MC4R gene in poultry.

• The Journal of the Korean Society for Microbiology
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• v.35 no.4
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• pp.283-288
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• 2000

The PCR primer set JW21-JW22 of Weiss et al. (19), which was reported to amplify a 139-bp fragment of the l6S rRNA gene of Helicobacter pylori, has been recently used for the detection of H. pylori in clinical specimens. However, when we applied JW21-JW22 PCR to other members of the genus Helicobacter and unrelated microorganisms, all of these bacteria produced a 139-bp PCR product. Therefore, we designed a novel primer set, HPU185-HPL826, which produced a 642-bp amplicon of the l6S rRNA gene of H. pylori. Then we further examined the specificity of the novel PCR assay using Southern blot hybridization with an internal probe, HPP225. The PCR assay described in this study was shown to be highly sensitive and specific only to the H. pylori 16S rRNA gene sequences.

• Journal of Animal Environmental Science
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• v.21 no.3
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• pp.93-98
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• 2015

The objective of this study was to review application of next-generation sequencing (NGS) to investigate microbiome in the livestock sector. Since the 16S rRNA gene is used as a phylogenetic marker, unculturable members of microbiome in nature or managed environments have been investigated using the NGS technique based on 16S rRNA genes. However, few NGS studies have been conducted to investigate microbiome in the livestock sector. The 16S rRNA gene sequences obtained from NGS are classified to microbial taxa against the 16S rRNA gene reference database such as RDP, Greengenes and Silva databases. The sequences also are clustered into species-level OTUs at 97% sequence similarity. Microbiome similarity among treatment groups is visualized using principal coordinates analysis, while microbiome shared among treatment groups is visualized using a venn diagram. The use of the NGS technique will contribute to elucidating roles of microbiome in the livestock sector.

• Korean Journal of Microbiology
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• v.47 no.3
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• pp.268-274
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• 2011

Anabaena (Cyanobacteria, Nostocales) are important for water quality controls, because they are often responsible for freshwater green tides; moreover, some species are reported to produce hepatotoxin. In this study, we sequenced RNA polymerase beta subunit (rpoB) gene of Anabaena, and evaluated their sequences for the potential use of a molecular taxonomic marker in this taxon. Anabaena rpoB showed low DNA similarity and high genetic divergences when compared those of 16S rRNA, and the molecular differences were statistically significant (Student t-test, p<0.01). Parsimony analyses showed the rpoB gene evolves 4.8-fold faster than 16S rRNA. In addition, phylogeny of the rpoB gene separated each Anabaena strain more clearly compared with a 16S rRNA tree. These results suggest that the rpoB gene is a useful marker for the molecular phylogenetics and the species discrimination of Anabaena.

• BMB Reports
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• v.38 no.1
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• pp.41-48
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• 2005

In this study we developed the transposon-mediated shuttle vector 'Hanpvid', which composed of HaNPV (Heliothis armigera nuclear polyhedrosis virus) genomic DNA and a transposon cassette from Bacmid of Bac-to-Bac system. Hanpvid replicates in E. coli in the same way as Bacmid and retains infective function in cotton bollworm cells (Hz-AM1). Using Hanpvid we constructed a recombinant virus, which could infect Hz-AM1 cells and generate recombinant HaNPV (rHa-Bar) containing the barnase gene, a ribonuclease gene from Bacillus amyloliquefaciens. Since the expression vector carrying barnase gene cannot replicate in the absence of barstar, a specific inhibitor of barnase, we constructed a new cotton bollworm cell line (AM1-NB) using the marker rescue method. In AM1-NB barstar was integrated into the cellular chromosome to sustain the replication of rHa-Bar. To screen out recombinant HaNPV for potential use as biopesticide, Hz-AM1 and AM1-NB cell lines were infected with rHa-Bar, respectively. The results obtained indicate that Viral progenies in AM1-NB were 23 and 160 times greater than those in Hz-AM1 48 h and 72 h after infection, respectively. With additional insertion of the polyhedron gene from AcNPV (Autographa californica nuclear polyhedrosis virus) into the Hanpvid genome, rHa-Bar regained the polyhedron phenotype and its pest-killing rate greatly improved. Toxic analysis showed that the lethal dosages ($LD_{50}$) and the lethal time(s) ($LT_{50}$) of rHa-Bar were reduced by 20% and 30%, respectively, compared to wt-HaNPV in the third instar larvae of cotton bollworm. This study shows that in AM1-NB barnase can be effectively produced and used as pest-killing agent for the biological control of cotton pests.

• KSBB Journal
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• v.16 no.6
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• pp.579-591
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• 2001

The cell growth inhibitor effect of cervical cancer cells was investigated by liposome mediated transfection (pRcCMVp53/lipofectin) and by transfection using adenovirus (AdCMVp57). The papilloma virus cancer cell lines we used in this study were HPV16 positive, having inhibiter gene, wild p53 gene, CaSki, SiHa, HPV18 positive HeLa, HeLaS3 and HPV negative C33A, HT3. LacZ gene of E.coli was used as the marker gene for the transfection efficiency. The effect on the inhibition of tumor cell growth was measured by cell count and cell viability though ELISA analysis and MTT assay. The inhibition of tumor cell growth was confirmed by measuring each assay for six days, comparing with the normal control cell growth. The cell growth of cervical cancer calls by transfection was significantly reduced and showed tittle differences among the cell lines. To eliminate the potential problem of Ad(adenovirus) contamination during rAAV production, rAAV can be produced by a triple transfection of vector plasmic, packaging plasmid, and adenovirus helper plasmid. To examine the helper functions of Ad plasmids on the production of rAAV vector, we carried out cotransfection of three plasmids, AAV vector, packaging construct, and Ad helper plasmids. The optimized transfection condition for calcium phosphate method is 25ug of total DNA per 10-cm-diameter plate of 293 cell. We found that rAAV yields peaked at 48hr after Ad infection. The titer of rAAV was measured by the dot blot analysis to measure the number of particles/ml based on the quantification of viral DNA. Recent1y, Kombucha(fungi) was identified as a very potent antileukefic agent. In the present study, effect of natural toxin(plankton) and Kombucha is PSP(GTXI-3, neoSTX), on various MTT assay cervical cancer cell line. Toxin(GTX 1-3, neoSTX) also inhibited the proliferation in primary cervical cancer calls in a dose-dependent toxin concentration. These results showed that toxin was very potent in inhibiting the proliferation of cervical cancer calls in vitro. Toxins and Kombuoha exhibited a dose dependent inhibition of cellular proliferation in cancer cell line.

• Korean Journal of Microbiology
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• v.44 no.3
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• pp.251-257
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• 2008

Four potential microcystin-producing cyanobacteria were isolated from large reservoirs which act as sources of drinking water supply in Korea. Strain DC-2, YD-l, and YD-6 were closely related to Microcystis aeruginosa based on the analysis of l6S rRNA gene and mcyA gene sequences. mcyA gene sequence of YDS2-3, isolated from Yongdam Reservoir, was closed to that of M. aeruginosa, whereas l6S rRNA gene sequence was not related to the known sequences of microcystin-producing cyanobacteria indicating this strain can be a novel cyanobacterium belonging to the genus Microcystis. When mcyA gene sequences of isolated cyanobacteria were compared with the mcyA gene sequence library of two reservoirs, the sequence of DC-2 matched with the dominant ones.

• Journal of Life Science
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• v.13 no.6
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• pp.856-864
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• 2003

The 16S rRNA gene is the most common gene in the phylogenetic analysis of procaryotes. However very high conservative of 16S rRNA has limitation in the discrimination of highly related organisms, hence other molecule was applied in this study and the result was compared with that of 16S rRNA. Three COGs (Clusters of Orthologous of protein) were only detected in 42 procaryotes ; transcription elongation facto. (COG0195), bacterial DNA primase (COG0358) and uridylate kinase (COG0528). Uridylate kinase gene was selected because of the similarity and one single copy number in each genome. Bacteria, belong to same genus, and Archaebacteria were same position with high bootstrap value in phylogenetic tree like the tree of 16S rRNA. However, alpha and epsilon Proteobcteria showed different position and Spirochaetales of Eubarteria was grouped together with Archaebacteria unlike the result of 16S rRNA. Uridylate kinase may compensate the problem of very high conservative of 16S rRNA gene and it would help to access more accurate discrimination and phylogenetic analysis of bacteria.

• Archives of Pharmacal Research
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• v.27 no.1
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• pp.111-117
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• 2004

Hepatic microcirculatory failure is a major component of reperfusion injury in the liver. Recent data provided some evidence that endothelium-derived vasoconstrictors and vasodilators may be functionally important to the control of the total hepatic blood flow under these conditions of circulatory failure. Since Kupffer cells provide signals that regulate the hepatic response in ischemia/reperfusion (I/R), the aim of this study was to investigate the role of Kupffer cells in the I/R-induced imbalance of vasoregulatory gene expression. Rats were subjected to 60 min hepatic ischemia, followed by 5 h of reperfusion. The Kupffer cells were inactivated by gadolinium chloride ($GdCl_3$, 7.5 mg/kg body weight, intravenously) 1 day prior to ischemia. Liver samples were obtained 5 hrs after reperfusion for RT-PCR analysis of the mRNA for genes of interest: endothelin-1 (ET-1), its receptors $ET_A and ET_B$, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1). ET-1 mRNA expression was increased by I/R. mRNA levels for $ET_A$ receptors showed no change, whereas $ET_B$ receptor transcripts increased in the I/R group. The increases in ET-1 and $ET_B$ mRNA were not prevented by the $GdCI_3$ pretreatment. The mRNA levels for iNOS and eNOS significantly increased within the I/R group with no significant difference between the I/R group and the $GdCl_3$-treated I/R group. HO-1 mRNA expression significantly increased in the I/R group and this increase was attenuated by $GdCI_3$. In conclusion, we have demonstrated that an imbalance in hepatic vasoregulatory gene expression occurs during I/R. Our findings suggest that the activation of Kupffer cells is not required for I/R-induced hepatic microvascular dysfunction.