A set of epitope-tagging integration vectors for functional analysis in <i>Saccharomyces cerevisiae</i>

Sung, Hyeran,Chul Han, Kyung,Chul Kim, Jun,Wan Oh, Ki,Su Yoo, Hwan,Tae Hong, Jin,Bok Chung, Youn,Lee, Chong-kil,Lee, Kyung S.,Song, Sukgil ELSEVIER 2005 FEMS YEAST RESEARCH Vol.5 No.10

<P><B>Abstract</B></P><P>Functional analysis of genes from <I>Saccharomyces cerevisiae</I> has been the major goal after determination of genome sequences. Even though several tools for molecular-genetic analyses have been developed, only a limited number of reliable genetic tools are available to support functional assay at protein level. Epitope tagging is a powerful tool for detecting, purifying, and functional studying of proteins. But systematic tagging systems developed with integration vectors are not available. Here, we have constructed a set of integration vectors allowing a translational fusion of interested proteins to the four different epitope tags (HA, Myc, Flag, and GFP). To confirm function and expression of C-terminal-tagged proteins, we used Cdc11, a component of the septin filament that encircles the mother bud neck and consists of five major proteins: Cdc3, Cdc10, Cdc11, Cdc12, and Sep7. The tagged version of Cdc11 expressed under its endogenous promoter was found to be physiologically functional, as evidenced by localization at the neck and suppression of the growth defect associated with the temperature-sensitive mutation of <I>cdc11-6</I>. The expressed proteins were efficiently detected with antibodies against Cdc11 or the epitopes. When immunoprecipitated with anti-Myc antibody, each septin protein tagged with Myc was effectively copurified with other septin components, indicating formation of a stable septin complex. Because the modules of the tags were located under the same array of eighteen restriction sites on integration vectors containing four different markers (<I>HIS3</I>, <I>TRP1</I>, <I>LEU2</I>, or <I>URA3</I>), this tagging system provides efficient multiple tagging and stable expression of a gene of interest.</P>

PCR-Based Detection of Mycoplasma Species

Sung Hyeran,Kang Seung Hye,Bae Yoon Jin,Hong Jin Tae,Chung Youn Bok,Lee Chong-Kil,Song Sukgil The Microbiological Society of Korea 2006 The journal of microbiology Vol.44 No.1

In this study, we describe our newly-developed sensitive two-stage PCR procedure for the detection of 13 common mycoplasmal contaminants (M. arthritidis, M. bovis, M. fermentans, M. genitalium, M. hominis, M. hyorhinis, M. neurolyticum, M. orale, M. pirum, M. pneumoniae, M. pulmonis, M. salivarium, U. urealyticum). For primary amplification, the DNA regions encompassing the 16S and 23S rRNA genes of 13 species were targeted using general mycoplasma primers. The primary PCR products were then subjected to secondary nested PCR, using two different primer pair sets, designed via the multiple alignment of nucleotide sequences obtained from the 13 mycoplasmal species. The nested PCR, which generated DNA fragments of 165-353 bp, was found to be able to detect 1-2 copies of the target DNA, and evidenced no cross-reactivity with the generated DNA of related microorganisms or of human cell lines, thereby confirming the sensitivity and specificity of the primers used. The identification of contaminated species was' achieved via the performance of restriction fragment length polymorphism (RFLP) coupled with Sau3AI digestion. The results obtained in this study furnish evidence suggesting that the employed assay system constitutes an effective tool for the disagnosis of mycoplasmal contamination in cell culture systems.

Phosphorylation-Dependent Septin Interaction of Bni5 is Important for Cytokinesis

Sung Chang Nam,Hyeran Sung,Seung Hye Kang,Jin Young Joo,이수재,정연복,이종길,송석길 한국미생물학회 2007 The journal of microbiology Vol.45 No.3

In budding yeast, septin plays as a scaffold to recruits protein components and regulates crucial cellular events including bud site selection, bud morphogenesis, Cdc28 activation pathway, and cytokinesis. Phosphorylation of Bni5 isolated as a suppressor for septin defect is essential to Swe1-dependent regulation of bud morphogenesis and mitotic entry. The mechanism by which Bni5 regulates normal septin function is not completely understood. Here, we provide evidence that Bni5 phosphorylation is important for interaction with septin component Cdc11 and for timely delocalization from septin filament at late mitosis. Phosphorylation-deficient bni5-4A was synthetically lethal with hof1Δ . bni5-4A cells had defective structure of septin ring and connected cell morphology, indicative of defects in cytokinesis. Two-hybrid analysis revealed that bni5-4A has a defect in direct interaction with Cdc11 and Cdc12. GFP-tagged bni5-4A was normally localized at mother-bud neck of budded cells before middle of mitosis. In contrast, at large-budded telophase cells, bni5-4A-GFP was defective in localization and disappeared from the neck approximately 2 min earlier than that of wild type, as evidenced by time-lapse analysis. Therefore, earlier delocalization of bni5-4A from septin filament is consistent with phosphorylation-dependent interaction with the septin component. These results suggest that timely de localization of Bni5 by phosphorylation is important for septin function and regulation of cytokinesis. In budding yeast, septin plays as a scaffold to recruits protein components and regulates crucial cellular events including bud site selection, bud morphogenesis, Cdc28 activation pathway, and cytokinesis. Phosphorylation of Bni5 isolated as a suppressor for septin defect is essential to Swe1-dependent regulation of bud morphogenesis and mitotic entry. The mechanism by which Bni5 regulates normal septin function is not completely understood. Here, we provide evidence that Bni5 phosphorylation is important for interaction with septin component Cdc11 and for timely delocalization from septin filament at late mitosis. Phosphorylation-deficient bni5-4A was synthetically lethal with hof1Δ. bni5-4A cells had defective structure of septin ring and connected cell morphology, indicative of defects in cytokinesis. Two-hybrid analysis revealed that bni5-4A has a defect in direct interaction with Cdc11 and Cdc12. GFP-tagged bni5-4A was normally localized at mother-bud neck of budded cells before middle of mitosis. In contrast, at large-budded telophase cells, bni5-4A-GFP was defective in localization and disappeared from the neck approximately 2 min earlier than that of wild type, as evidenced by time-lapse analysis. Therefore, earlier delocalization of bni5-4A from septin filament is consistent with phosphorylation-dependent interaction with the septin component. These results suggest that timely delocalization of Bni5 by phosphorylation is important for septin function and regulation of cytokinesis.

Requirement of Bni5 Phosphorylation for Bud Morphogenesis in Saccharomyces cerevisiae

Sung Chang Nam,Hyeran Sung,정연복,이종길,이동훈,송석길 한국미생물학회 2007 The journal of microbiology Vol.45 No.1

In budding yeast, G2/M transition is tightly correlated with bud morphogenesis regulated by Swe1 and septin that plays as a scaffold to recruits protein components. BNI5 isolated as a suppressor for septin defect is implicated in septin organization and cytokinesis. The mechanism by which Bni5 regulates normal septin function is not completely understood. Here, we show that Bni5 phosphorylation is required for mitotic entry regulated by Swe1 pathway. Bni5 modification was evident from late mitosis to G1 phase, and CIP treatment in vitro of affinity-purified Bni5 removed the modification, indicative of phosphorylation on Bni5. The phosphorylation-deficient mutant of BNI5 (bni5-4A) was defective in both growth at semi-restrictive temperature and suppression of septin defect. Loss of Bni5 phosphorylation resulted in abnormal bud morphology and cell cycle delay at G2 phase, as evidenced by the formation of elongated cells with multinuclei. However, deletion of Swe1 completely eliminated the elongated-bud phenotypes of both bni5 deletion and bni5-4A mutants. These results suggest that the bud morphogenesis and mitotic entry are positively regulated by phosphorylation-dependent function of Bni5 which is under the control of Swe1 morphogenesis pathway.

결장암에 대한 활성 자연살해세포의 항암효능

성혜란(Hyeran Sung),김지연(Jee Youn Kim),박민경(Min Gyeong Park),김일회(Il-Hoi Kim),이동욱(Dong Wook Lee),한상배(Sang-Bae Han),이종길(Chong-Kil Lee),송석길(Sukgil Song) 대한약학회 2010 약학회지 Vol.54 No.3

Colorectal cancer is one of the most common alimentary malignancies. In this study, the antitumor activity of activated human natural killer (NK) cells against human colorectal cancer was evaluated in vivo. Human NK cells are the key contributors of innate immune response and the effective functions of these cells are enhanced by cytokines. Human peripheral blood mononuclear cells (PBMC) were cultured with interleukin-2 (IL-2)-containing medium for 14 days and resulted in enriched NK cell population. The resulting populations of the cells comprised 7% CD3+CD4+ cells, 25% CD3+CD8+ cells, 13% CD3-CD8+ cells, 4% CD3+CD16/CD56+ cells, 39% CD3+CD16/CD56- cells, and 52% CD3-CD16/CD56+ cells. Tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), IL-2, IL-4, and IL-5 transcripts of the activated NK cells were confirmed by RT-PCR. In addition, activated NK cells at doses of 2.5, 5 and 10 million cells per mouse inhibited 10%, 34% and 47% of SW620-induced tumor growth in nude mouse xenograft assays, respectively. This study suggests that NK cell-based immunotherapy may be used as an adoptive immunotherapy for colorectal cancer patients.

Inhibitory Effect of Natural Killer Cells on Liver Tumor Growth in Mouse Xenograft Model

Sukgil Song, Ji-sung Park, Hyeran Sung, Il-Hoi Kim, Chong-Kil Lee, Jin Tae Hong 충북대학교 동물의학연구소 2012 Journal of Biomedical and Translational Research Vol.13 No.2

Human natural killer (NK) cells are major players in innate immune response. The functions of these cells as a scavenger of cancer cells are enhanced by cytokines such as interleukin-2 (IL-2), which play an important role in immune response in both tumors and virally infected cells. Liver cancer has a high incidence rate and is a major cause of death in Korea. We provide evidence that human NK cells inhibit tumor growth of the hepatocellular carcinoma cell line SNU-354. NK cells were cultured with human IL-2 for 14 days, yielding an enriched NK cell population containing 35% CD8+cells, 6% CD4+cells, and 51% CD16+/CD56+ cells. Intravenous injection of NK cells at doses from 2.5 to 10 million cells/mouse was administered once per week in a nude mouse model that retains human liver tumor induced by implantation of SNU-354 cells. The results showed that human NK cells were recruited within tumor tissue and inhibited SNU-354 tumor growth by 32%, 58%, and 65%. The current data suggest the potential for use of NK cellbased immunotherapy for treatment of human liver cancer.

[S3-1] Nutrients and DNA Methylation

Hyeran Jang,Sang-Woon Choi 한국식품영양과학회 2007 한국식품영양과학회 학술대회발표집 Vol.2007 No.10

Targeted Genome Editing for Crop Improvement

( Hyeran Kim ),( Sang Tae Kim ),( Sang Gyu Kim ),( Jin Soo Kim ) 한국육종학회 2015 Plant Breeding and Biotechnology Vol.3 No.4

Crop improvement is essential to attaining world food security and enhancing nutrition for human beings. Both conventional breeding and modern molecular breeding have contributed to increased crop production and quality. However, the time and resources for breeding practices have been limited. It takes a long time to bring a novel improved crop to the market, and the genetic sources from wild species cannot be always available for crops of our interests. Genome editing technology implemented molecular breeding can overcome those limitations of time and resource by facilitating the specific editing of plant genomes. However, there is a long-lasting argument about the safety of genetically modified organisms (GMOs). In this review, we briefly summarize the principle of genome editing tools, focusing on the CRISPR/Cas9 system and the application of these tools to plants in the service of crop engineering.

WTC in EMI: Correlational Factors and College Learners' Perceptions

Hyeran Lee,Kiwan Sung 한국외국어교육학회 2014 Foreign languages education Vol.21 No.2

This paper investigates the differences in college learners' perceptions and correlations of factors related to willingness to communicate (WTC) in an English mediated instruction (EMI) class. Based on a survey of 50 collegiates (33 males, 17 females) and two rounds of interviews with 15 students, the t-tests showed that there were not many statistical differences depending on learner characteristics except the learners' grade difference affecting their perception of improvement in English. However, depending on the course types, the Kruskal-Wallis test showed statistically significant differences in the categories of present level of participation, expected level of participation with L1 option, question & answer, group collaboration, active listening, and preference to EMI with L1 option. Furthermore, the factors of WTC appeared strongly correlated with their perceptions on the improvement in English, increased confidence, and the extent of learning in the EMI class. According to qualitative analyses of open-ended questions in the survey and interviews, the learners thought group presentations in English were most difficult. They also responded that their low English proficiency, peer pressure, and the student-orientedness in class made them passive and less confident. The learners, however, adopted diverse coping strategies to overcome such difficulties. They were also positive about the limited use of L1 in the EMI class. Implications for EMI are suggested.

Peroxiredoxin-2 Upregulated by NF-κB Attenuates Oxidative Stress During the Differentiation of Muscle-Derived C2C12 Cells

Won, Hyeran,Lim, Sangbin,Jang, Miran,Kim, Yeonghwan,Rashid, Md Abdur,Jyothi, K.R.,Dashdorj, Amarjargal,Kang, Insug,Ha, Joohun,Kim, Sung Soo Mary Ann Liebert 2012 ANTIOXIDANTS AND REDOX SIGNALING Vol.16 No.3