• Korean Journal of Poultry Science
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• v.41 no.4
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• pp.287-296
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• 2014

Chicken eggs undergo various physiological changes during egg maturation. To study genes associated with the egg maturation in pre-ovulation (immature) and post-ovulation (mature), we compared gene expression patterns between in the immature egg and mature egg using RNA sequencing data. Mature and immature eggs were obtained from a Heuksaek Jaerae-jong of Korean native chicken. Total RNAs obtained from the eggs were sequenced by Illumina HiSeq 2000 platform, and the generated sequence reads were mapped to Galgal4 reference sequence assembly using Tuxedo Protocol. From the comparison of the RNA sequencing data, 315 genes were differentially expressed between mature and immature eggs, and 46 genes were only detected in immature egg. Further gene ontology (GO) analysis was performed for the differentially expressed genes using DAVID, showing that 29 and 28 GO terms were independently clustered from mature and immature, respectively. From those clustered GO terms, genes related to germ cell development, sex differentiation and defense response to bacterium were mainly expressed in the immature egg, while genes related to regulation of apoptosis, steroid metabolic process and lipid homeostasis were mainly detected in the mature egg. Our results could contribute to understand egg maturation before and after ovulation, and develop genetic markers for improving egg quality and productivity.

• Journal of Food Hygiene and Safety
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• v.30 no.3
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• pp.281-288
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• 2015

Vibrio is a genus of Gram-negative, curved, halophilic, and non-spore-forming bacteria. Some of the Vibrio species, such as V. cholerae and V. parahaemolyticus, often contaminate seafood products and occasionally cause human diseases when the seafood products are ingested. A total of 24 Vibrio strains were isolated from shrimp samples on Thiosulphate citrate bile salt sucrose (TCBS) media in this study. All of the 24 isolates were confirmed to belong to the genus Vibrio by using 16S rRNA gene sequence analyses. Vitek 2 system and species-specific polymerase chain reaction (PCR) methods were used to further identify a total of 29 Vibrio strains at the species level, including the 24 shrimp Vibrio isolates and five Vibrio reference strains. The specificities of the two methods to identify Vibrio strains at the species level were compared in this study. The species-specific PCR method was designed to detect five different Vibrio species, such as Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, and Vibrio mimicus. From the 24 Vibrio shrimp isolates, the Vitek 2 system method could identify 15 (62.5%) strains as Vibrio species and 7 (29.2%) strains as non-Vibrio species, but could not identify the rest 2 (8.3%) strains. But species-specific PCR method could identify 16 (66.7%) strains as Vibrio species and could not identify the rest 8 (33.3%) strains. Among the 24 Vibrio shrimp strains, these two methods could unanimously identify 7 (7/24, 29.2%) strains (2 V. parahaemolyticus, 4 V. alginolyticus, and 1 V. mimicus). Considering that such different identification results were obtained using the two different methods in this study, identification method for Vibrio species must be carefully chosen.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.67 no.1
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• pp.41-52
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• 2022

As a cultivar of Korean wheat, 'Keumgang' wheat variety has a fast growth period and can be grown stably. Hexaploid wheat (Triticum aestivum) has moderately high salt tolerance compared to tetraploid wheat (Triticum turgidum L.). However, the molecular mechanisms related to salt tolerance of hexaploid wheat have not been elucidated yet. In this study, the candidate genes related to salt tolerance were identified by investigating the genes that are differently expressed in Keumgang variety and examining salt tolerant mutation '2020-s1340.'. A total of 85,771,537 reads were obtained after quality filtering using NextSeq 500 Illumina sequencing technology. A total of 23,634,438 reads were aligned with the NCBI Campala Lr22a pseudomolecule v5 reference genome (Triticum aestivum). A total of 282 differentially expressed genes (DEGs) were identified in the two Triticum aestivum materials. These DEGs have functions, including salt tolerance related traits such as 'wall-associated receptor kinase-like 8', 'cytochrome P450', '6-phosphofructokinase 2'. In addition, the identified DEGs were classified into three categories, including biological process, molecular function, cellular component using gene ontology analysis. These DEGs were enriched significantly for terms such as the 'copper ion transport', 'oxidation-reduction process', 'alternative oxidase activity'. These results, which were obtained using RNA-seq analysis, will improve our understanding of salt tolerance of wheat. Moreover, this study will be a useful resource for breeding wheat varieties with improved salt tolerance using molecular breeding technology.

• Genomics & Informatics
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• v.10 no.1
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• pp.1-8
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• 2012

Recently, the technologies of DNA sequence variation and gene expression profiling have been used widely as approaches in the expertise of genome biology and genetics. The application to genome study has been particularly developed with the introduction of the nextgeneration DNA sequencer (NGS) Roche/454 and Illumina/ Solexa systems, along with bioinformation analysis technologies of whole-genome $de$ $novo$ assembly, expression profiling, DNA variation discovery, and genotyping. Both massive whole-genome shotgun paired-end sequencing and mate paired-end sequencing data are important steps for constructing $de$ $novo$ assembly of novel genome sequencing data. It is necessary to have DNA sequence information from a multiplatform NGS with at least $2{\times}$ and $30{\times}$ depth sequence of genome coverage using Roche/454 and Illumina/Solexa, respectively, for effective an way of de novo assembly. Massive shortlength reading data from the Illumina/Solexa system is enough to discover DNA variation, resulting in reducing the cost of DNA sequencing. Whole-genome expression profile data are useful to approach genome system biology with quantification of expressed RNAs from a wholegenome transcriptome, depending on the tissue samples. The hybrid mRNA sequences from Rohce/454 and Illumina/Solexa are more powerful to find novel genes through $de$ $novo$ assembly in any whole-genome sequenced species. The $20{\times}$ and $50{\times}$ coverage of the estimated transcriptome sequences using Roche/454 and Illumina/Solexa, respectively, is effective to create novel expressed reference sequences. However, only an average $30{\times}$ coverage of a transcriptome with short read sequences of Illumina/Solexa is enough to check expression quantification, compared to the reference expressed sequence tag sequence.

• The Journal of the Korean Society for Microbiology
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• v.35 no.3
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• pp.263-271
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• 2000

A clinical isolate of Klebsiella pneumoniae K7746 produced the extended-spectrum ${\beta}$-lactamase (ESBL) SHV-12. A 6.6 kb BamHI fragment containing the $bla_{SHV-12}$ gene of K7746 strain was cloned into pCRScriptCAM vector resulting in the recombinant plasmid p7746-Cl. The restriction map of 3.6 kb inserted DNA and sequences immediately surrounding $bla_{SHV-12}$ of p7746-C1 were homologous to plasmid pMPA2a carrying $bla_{SHV-2a}$. In addition, both $bla_{SHV-12}$ and $bla_{SHV-2a}$ were expressed from a common hybrid promoter made of the -35 region derived from the left inverted repeat of IS26 and the -10 region from the $bla_{SHV}$ promoter itself. The results indicate that $bla_{SHV-12}$ and $bla_{SHV-2a}$ may have evolved from a common ancestor in the sequential order of $bla_{SHV-2a}$ first, followed by $bla_{SHV-12}$. Furthermore, by the PCR mapping method using primers corresponding to the IS26 and $bla_{SHV}$, the association between IS26 and $bla_{SHV}$ was studied in 12 clinical isolates carrying $bla_{SHV-2a}$, 27 clinical isolates carrying $bla_{SHV-12}$, and 5 reference strains carrying $bla_{SHV-1}$ to $bla_{SHV-5}$. All 39 strains carrying $bla_{SHV-2a}$ or $bla_{SHV-12}$ were positive by the PCR, providing confirmative evidence that IS26 has been involved in the evolution and dissemination of $bla_{SHV-2a}$ and $bla_{SHV-12}$. But 5 reference strains carrying $bla_{SHV-1}$ to $bla_{SHV-5}$ were negative by the PCR. Therefore, we concluded that the molecular evolutionary pathway of $bla_{SHV-2a}$ and $bla_{SHV-12}$ may be different from that of other $bla_{SHV-ESBL}$, e.g., $bla_{SHV-2}$, $bla_{SHV-3}$, $bla_{SHV-4}$, and $bla_{SHV-5}$.

• Journal of The Korean Society of Inherited Metabolic disease
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• v.15 no.1
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• pp.44-48
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• 2015

Mucopolysaccharidosis (MPS) IIIA is a lysosomal storage disorder caused by abnormalities of the enzyme Heparan N-sulfatase that is required for degradation of heparan sulfate. The patient in this study was a 5 year-old boy who presented with macrocephaly and developmental delay. Urinary excretion of glycosaminoglycan was increased (26 g/moL creatinine, reference range: <7 g/moL creatinine) and a distinct band of heparan sulfate was shown in electrophoresis. Heparan N-sulfatase activity was significantly decreased in skin fibroblasts (0.2 pmoL/min/mg protein, reference range: 9-64 pmoL/min/mg protein). PCR and direct sequencing analysis of the SGSH gene showed compound heterozygous mutations: c.1040C>T (p.S347F) and c.703G>A (p.D235N). This is the first report for a Korean patient with MPS IIIA who was confirmed by biochemical investigation and molecular genetic analyses.

• Nutrition Research and Practice
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• v.13 no.1
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• pp.58-63
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• 2019

BACKGROUND/OBJECTIVES: Telomeres are located at the chromosomal ends and progressively shortened during each cell cycle. Telomerase, which is regulated by hTERT and c-MYC, maintains telomeric DNA sequences. Especially, telomerase is active in cancer and stem cells to maintain telomere length for replicative immortality. Recently we reported that walnut phenolic extract (WPE) can reduce cell viability in a colon cancer stem cell (CSC) model. We, therefore, investigated the effect of WPE on telomere maintenance in the same model. MATERIALS AND METHODS: $CD133^+CD44^+$ cells from HCT116, a human colon cancer cell line, were sorted by Fluorescence-activated cell sorting (FACS) and treated with WPE at the concentrations of 0, 10, 20, and $40{\mu}g/mL$ for 6 days. Telomere lengths were assessed by quantitative real-time PCR (qRT-PCR) using telomere specific primers and DNA extracted from the cells, which was further adjusted with single-copy gene and reference DNA ($ddC_t$). Telomerase activity was also measured by qRT-PCR after incubating the PCR mixture with cell protein extracts, which was adjusted with reference DNA ($dC_t$). Transcriptions of hTERT and c-MYC were determined using conventional RT-PCR. RESULTS: Telomere length of WPE-treated cells was significantly decreased in a dose-dependent manner ($5.16{\pm}0.13$ at $0{\mu}g/mL$, $4.79{\pm}0.12$ at $10{\mu}g/mL$, $3.24{\pm}0.08$ at $20{\mu}g/mL$ and $3.99{\pm}0.09$ at $40{\mu}g/mL$; P = 0.0276). Telomerase activities concurrently decreased with telomere length ($1.47{\pm}0.04$, $1.09{\pm}0.01$, $0.76{\pm}0.08$, and $0.88{\pm}0.06$; P = 0.0067). There was a positive correlation between telomere length and telomerase activity (r = 0.9090; P < 0.0001). Transcriptions of both hTERT and c-MYC were also significantly decreased in the same manner. CONCLUSION: In the present cell culture model, WPE reduced telomere maintenance, which may provide a mechanistic link to the effect of walnuts on the viability of colon CSCs.

• Journal of fish pathology
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• v.20 no.3
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• pp.201-209
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• 2007

Viral hemorrhagic septicemia (VHS) is one of the most serious viral disease of farmed rainbow trout and some marine fishes in Europe and North America. It has been reported in various marine fish species of Asian countries and induced cause mass mortality in Japanese flounder (Paralichthys olivaceus) culturing in Korea. The aims of this study were to monitor VHSV in wild marine fishes and to give critical information for controling the disease through prophylactic methods. Prevalence of the viral disease, geological distribution and reservoir of the virus were investigated using wild marine fishes captured in southern coast and east china sea for two years. (Reverse Transcriptase Polymerase Chain Reaction) RT-PCR results showed that VHSV were detected in 17 (10.6%) out of 160 fish. G gene sequences of viral strains isolated in this study were closely related to that of a reference strain, KVHS01-1, belonging to VHSV genotype Ⅰ. The results suggest that some of wild marine fishes are VHSV carriers and may spread the pathogen directly to fish farmed in coastal area.

• Biomedical Science Letters
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• v.4 no.1
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• pp.43-56
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• 1998

A multiplex PCR method was designed by employing primers specific for the eaeA gene, conserved sequences of Shiga-like toxins (SLT-I.II), and the 60-MDa plasmid of enterohemorrhagic E. coli (EHEC) O157:H7 strain. A set of six synthetic oligonucleotide primers derived from sequences of the SLT-I.II, eaeA, and 60-MDa plasmid genes of E. coli O157:H7 were used in a multiplex PCR amplification procedure to detect these genes in the same enteric pathogens. In two enterohemorrhagic E. coli O157:H7 (ATCC 35150, ATCC 43894) reference strains, PCR products of 317bps (eaeA), 228bps (SLT-I.II), and 167bps (60-MDa plasmid) were successfully amplified simultaneously in a single reaction. However, the specific PCR products were not amplified in control strains of other enteric bacteria. The sensitivity of the multiplex PCR assay for detection of the SLT-I.II, eaeA, and 60-MDa plasmid genes of E. coli O157:H7 was found to be 2.5$\times$10$^{6}$ of bacteria in diarrheal stool to amplify all three bands. The multiplex PCR technology will allow large-scale screening of many clinical specimens or contaminated foods, and will be a very useful method for the detection of a wide range of microorganisms present in the environment, including EHEC O157:H7 in various types of specimens. The multiplex PCR assay has the potential to be used as a specific and rapid method for clinical diagnosis of disease caused by EHEC O157:H7.

• Journal of Plant Biotechnology
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• v.44 no.4
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• pp.372-378
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• 2017

For comparison of the transcription profiles in apple (Malus domestica L.) cultivars differing in flesh color expression, two cDNA libraries were constructed. Differences in gene expression between red flesh apple cultivar, 'Redfield' and white flesh apple cultivar, 'Granny Smith' were investigated by next-generation sequencing (NGS). Expressed sequence tag (EST) of clones from the red flesh apple cultivar and white flesh apple cultivar were selected for nucleotide sequence determination and homology searches. High resolution melting (HRM) technique measures temperature induced strand separation of short PCR amplicons, and is able to detect variation as small as one base difference between red flesh apple cultivars and white flesh apple cultivars. We applied high resolution melting (HRM) analysis to discover single nucleotide polymorphisms (SNP) based on the predicted SNP information derived from the apple EST database. All 103 pairs of SNPs were discriminated, and the HRM profiles of amplicons were established. Putative SNPs were screened from the apple EST contigs by HRM analysis displayed specific difference between 10 red flesh apple cultivars and 11 white flesh apple cultivars. In this study, we report an efficient method to develop SNP markers from an EST database with HRM analysis in apple. These SNP markers could be useful for apple marker assisted breeding and provide a good reference for relevant research on molecular mechanisms of color variation in apple cultivars.