• 미생물학회지
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• 제52권4호
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• pp.487-494
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• 2016

출아 효모에서의 Paf1 복합체는 총5개의 단백질로 구성되어있고, 구성성분들은 출아효모, 초파리, 식물들, 그리고 인간에 이르기까지 구조적으로, 기능적으로 잘 보존되어 있다. RNA 중합효소 II와 결합한 상태로 전사 개시부위부터 종결부위까지 함께 이동하며, 여러 전사인자들의 유입을 위한 매개체로 작용하여, 유전자 발현 조절의 핵심적인 역할을 수행한다. Paf1 복합체는 H2BK123 monoubiquitination에 기여하고, histone crosstalk에 의해 간접적으로 H3K4의 di-, tri-methylation에 기여하는 것이 알려져 있다. 하지지만, Paf1 복합체 구성요소들의 개별적인 기능에 대해서는 연구가 되어있지 않다. 이 연구에서는, Paf1 복합체 구성요소들의 단일 결핍 돌연변이 균주를 만든 후, 이들의 H2BK123 monoubiquitination 및 H3K4 mono-, di-, tri-methylation에 미치는 영향을 관찰했다. 놀랍게도, ${\Delta}paf1$, ${\Delta}rtf1$, ${\Delta}ctr9$ 돌연변이 균주에서는 H2Bub에 영향을 받는 H3K4me2와 H3K4me3뿐 아니라, H2B monoubiquitination에 영향을 받지 않는 H3K4 monomethylation의 심각한 감소를 관찰했다. 그러나, methyl기 전달 효소인 Set1의 발현 정도는 이 돌연변이 균주들에서 변하지 않았다. 이러한 결과로부터, Paf1 복합체가 Set1의 활성이나 Set1 복합체의 안정성을 직접 조절함으로써 H3K4 methylation을 조절할 수 있음을 제시한다.

• 한국양식학회지
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• 제17권1호
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• pp.1-7
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• 2004

A rainbow trout uncoupling protein 3 (UCP3) cDNA clone, encoding a 310 amino acid protein, was cloned and sequenced from a liver cDNA library. Two different splice variants designated UCP3-vl and UCP3-v2, were identified through liver cDNA library screening using rainbow trout UCP3 cDNA clone as a probe. UCP3-vl has 3 insertions in the UCP3 cDNA: the first insertion (133 bp), the second (141 bp), and the third (370 bp) were located 126 bp, 334 bp and 532 bp downstream from the start codon, respectively. UCP3-v2 contained a single insertion, identical in sequence and location to the second insertion of UCP3-vl. UCP3, a mitochondrial protein, functions to modulate the efficiency of oxidative phosphorylation. UCP3 has been detected from heart, testis, spinal cord, eye, retina, colon, muscle, brown adipose tissue and white adipose tissue in mammalian animals. Human and rodent UCP3s are highly expressed in skeletal muscle and brown adipose tissue, while they show weak expression of UCP3 in heart and white adipose tissue. In contrast to mammalian studies, RT-PCR and Southern blot analysis of the rainbow trout demonstrated that UCP3 is strongly expressed in liver and heart. UCP3, UCP3-vl, and UCP3-v2 all contain an Ava III short interspersed element (SINE), located in the 3'untraslated region (UTR). PCR using primers from the Ava III SINE and the UCP3 3'UTR region indicates that the UCP3 cDNA is structurally conserved among salmonids and that these primers may be useful for salmonid species genotyping.

• BMB Reports
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• 제39권5호
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• pp.502-510
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• 2006

2C-methyl-D-erythritol 2, 4-cyclodiphosphate synthase (MECPS, EC: 4.6.1.12) is the fifth enzyme of the non-mevalonate terpenoid pathway for isopentenyl diphosphate biosynthesis and is involved in the methylerythritol phosphate (MEP) pathway for ginkgolide biosynthesis. The full-length mecps cDNA sequence (designated as Gbmecps) was cloned and characterized for the first time from gymnosperm plant species, Ginkgo biloba, using RACE (rapid amplification of cDNA ends) technique. The full-length cDNA of Gbmecps was 874 bp containing a 720 bp open reading frame (ORF) encoding a peptide of 239 amino acids with a calculated molecular mass of 26.03 kDa and an isoelectric point of 8.83. Comparative and bioinformatic analyses revealed that GbMECPS showed extensive homology with MECPSs from other species and contained conserved residues owned by the MECPS protein family. Phylogenetic analysis indicated that GbMECPS was more ancient than other plant MECPSs. Tissue expression pattern analysis indicated that GbMECPS expressed the highest in roots, followed by in leaves, and the lowest in seeds. The color complementation assay indicated that GbMECPS could accelerate the accumulation of $\beta$-carotene. The cloning, characterization and functional analysis of GbMECPS will be helpful to understand more about the role of MECPS involved in the ginkgolides biosynthesis at the molecular level.

• Parasites, Hosts and Diseases
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• 제54권4호
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• pp.385-391
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• 2016

The discovery and understanding of antigenic proteins are essential for development of a vaccine against malaria. In Plasmodium falciparum, Pf92 have been characterized as a merozoite surface protein, and this protein is expressed at the late schizont stage, but no study of Pv92, the orthologue of Pf92 in P. vivax, has been reported. Thus, the protein structure of Pv92 was analyzed, and the gene sequence was aligned with that of other Plasmodium spp. using bioinformatics tools. The recombinant Pv92 protein was expressed and purified using bacterial expression system and used for immunization of mice to gain the polyclonal antibody and for evaluation of antigenicity by protein array. Also, the antibody against Pv92 was used for subcellular analysis by immunofluorescence assay. The Pv92 protein has a signal peptide and a sexual stage s48/45 domain, and the cysteine residues at the N-terminal of Pv92 were completely conserved. The N-terminal of Pv92 was successfully expressed as soluble form using a bacterial expression system. The antibody raised against Pv92 recognized the parasites and completely merged with PvMSP1-19, indicating that Pv92 was localized on the merozoite surface. Evaluation of the human humoral immune response to Pv92 indicated moderate antigenicity, with 65% sensitivity and 95% specificity by protein array. Taken together, the merozoite surface localization and antigenicity of Pv92 implicate that it might be involved in attachment and invasion of a merozoite to a new host cell or immune evasion during invasion process.

• 한국가금학회:학술대회논문집
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• 한국가금학회 2005년도 제22차 정기총회 및 학술발표회
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• pp.64-65
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• 2005

Pumilio 유전자는 생식 세포의 발달과 분화에 중요한 역할을 한다고 알려져 있다. 우리는 이러한 Pumilio family인 Pum1, Pum2 유전자를 닭에서 클로닝하여 그 Pumilio homology domain의 구조와 단백질 염기서열이 초파리, 생쥐, 인간과 유사하다는 것을 밝혔고 이를 통해 이 유전자가 진화적으로 보존되어 있다는 것을 증명하였다. 또한 닭의 Pum1과 Pum2 genome 구조 역시 생쥐와 인간 Pum 유전자들의 구조와 일치하는 것을 보여주었다. Real-time RT-PCR 결과 닭의 배아의 여러 조직들 중 Pum1과 Pum2 유전자 모두 부화한 암컷 생식선에서의 발현 수준이 유의적으로 높았고, 특히 Pum2 유전자의 경우 부화한 병아리의 생식선뿐만이 아니라 12일령의 생식선에서도 발현 수준이 높았다. 결과적으로, 다른 동물에서 알려진 바와 같이 닭에서도 Pumilio 유전자들이 생식선 발달에 관여할 가능성이 크다는 것을 알수 있다.

• Asian Pacific Journal of Cancer Prevention
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• 제17권3호
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• pp.1037-1047
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• 2016

HOX transcription factors are evolutionarily conserved in many different species and are involved in important cellular processes such as morphogenesis, differentiation, and proliferation. They have also recently been implicated in carcinogenesis, but their precise role in cancer, especially in cervical cancer (CC), remains unclear. In this work, using microarray assays followed by the quantitative polymerase chain reaction (qPCR), we found that the expression of 25 HOX genes was downregulated in CC derived cell lines compared with non-tumorigenic keratinocytes. In particular, the expression of HOXA9 was observed as down-modulated in CC-derived cell lines. The expression of HOXA9 has not been previously reported in CC, or in normal keratinocytes of the cervix. We found that normal CC from women without cervical lesions express HOXA9; in contrast, CC cell lines and samples of biopsies from women with CC showed significantly diminished HOXA9 expression. Furthermore, we found that methylation at the first exon of HOXA9 could play an important role in modulating the expression of this gene. Exogenous restoration of HOXA9 expression in CC cell lines decreased cell proliferation and migration, and induced an epithelial-like phenotype. Interestingly, the silencing of human papilloma virus (HPV) E6 and E7 oncogenes induced expression of HOXA9. In conclusion, controlling HOXA9 expression appears to be a necessary step during CC development. Further studies are needed to delineate the role of HOXA9 during malignant progression and to afford more insights into the relationship between downmodulation of HOXA9 and viral HPV oncoprotein expression during cercical cancer development.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2010년도 정기총회 및 춘계학술발표회
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• pp.17-17
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• 2010

Ethylene, known as a stress hormone regulate wide developmental processes including germination, root hair initiation, root and shoot primordial formation and elongation, leaf and flower senescence and abscission, fruit ripening. The acceleration of ethylene biosynthesis in plant associated with environmental and biological stresses. 1-Aminocycloprophane-1-carboxlyate deaminase(ACCD) is an enzyme that cleaves ACC into and ammonia, a precursor of the plant hormone ethylene. Plant growth-promoting rhizobacteria (PGPR) having ACCD can decrease endogenous ACC level of tissue, resulting in reduced production of ethylene in plants. ACC deaminse was a key enzyme for protect stressed plants from injurious effects of ethylene. ACCD gene was encoded from Pseudomonas flourescens, PGPR and was cloned in Escherichia coli. We expressed the recombinant ACCD(rACCD) containing 357 amino acids with molecular weight 39 kDa that revealed by SDS-PAGE and western blot. The rACCD was purified by Ni-NTA purification system. The active form of rACCD having enzyme activity converted ACC to a-ketobutyrate. The optimal pH for ACC deaminase activity was pH 8.5, but no activity below pH 7.0 and a less severe tapering activity at base condition resulting in loss of activity at over pH 11. The optimal temperature of the enzyme was $30^{\circ}$ and a slightly less severe tapering activity at 15 - 30$^{\circ}$, but no activity over $35^{\circ}$. P. flourescens ACC deaminase has a highly conserved residue that plays in allowing substrate accessibility to the active sites. The enzymatic properties of this rACCD will provide an important reference for analysis of newly isolated ACCD and identification of newly isolated PGPR containing ACCD.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2017년도 9th Asian Crop Science Association conference
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• pp.36-36
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• 2017

Out of twenty common protein amino acids, there are many kinds of non protein amino acids (NPAAs) that exist as secondary metabolites and exert ecological functions in plants. Mimosine (Mim), one of those NPAAs derived from L. leucocephala acts as an iron chelator and reversely block mammalian cell cycle at G1/S phases. Cysteine (Cys) is decisive for protein and glutathione that acts as an indispensable sulfur grantor for methionine and many other sulfur-containing secondary products. Cys biosynthesis includes consecutive two steps using two enzymes-serine acetyl transferase (SAT) and O-acetylserine (thiol)lyase (OASTL) and appeared in plant cytosol, chloroplast, and mitochondria. In the first step, the acetylation of the ${\beta}$-hydroxyl of L-serine by acetyl-CoA in the existence of SAT and finally, OASTL triggers ${\alpha}$, ${\beta}$-elimination of acetate from OAS and bind $H_2S$ to catalyze the synthesis of Cys. Mimosine synthase, one of the isozymes of the OASTLs, is able to synthesize Mim with 3-hydroxy-4-pyridone (3H4P) instead of $H_2S$ for Cys in the last step. Thus, the aim of this study was to clone and characterize the cytosolic (Cy) OASTL gene from L. leucocephala, express the recombinant OASTL in Escherichia coli, purify it, do enzyme kinetic analysis, perform docking dynamics simulation analysis between the receptor and the ligands and compare its performance between Cys and Mim synthesis. Cy-OASTL was obtained through both directional degenerate primers corresponding to conserved amino acid region among plant Cys synthase family and the purified protein was 34.3KDa. After cleaving the GST-tag, Cy-OASTL was observed to form mimosine with 3H4P and OAS. The optimum Cys and Mim reaction pH and temperature were 7.5 and $40^{\circ}C$, and 8.0 and $35^{\circ}C$ respectively. Michaelis constant (Km) values of OAS from Cys were higher than the OAS from Mim. Inter fragment interaction energy (IFIE) of substrate OAS-Cy-OASTL complex model showed that Lys, Thr81, Thr77 and Gln150 demonstrated higher attraction force for Cys but 3H4P-mimosine synthase-OAS intermediate complex showed that Gly230, Tyr227, Ala231, Gly228 and Gly232 might provide higher attraction energy for the Mim. It may be concluded that Cy-OASTL demonstrates a dual role in biosynthesis both Cys and Mim and extending the knowledge on the biochemical regulatory mechanism of mimosine and cysteine.

• Parasites, Hosts and Diseases
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• 제48권4호
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• pp.319-324
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• 2010

A family of calcium-dependent protein kinases (CDPKs) is a unique enzyme which plays crucial roles in intracellular calcium signaling in plants, algae, and protozoa. CDPKs of malaria parasites are known to be key regulators for stage-specific cellular responses to calcium, a widespread secondary messenger that controls the progression of the parasite. In our study, we identified a gene encoding Plasmodium vivax CDPK4 (PvCDPK4) and characterized its molecular property and cellular localization. PvCDPK4 was a typical CDPK which had well-conserved N-terminal kinase domain and C-terminal calmodulin-like structure with 4-EF hand motifs for calcium-binding. The recombinant protein of EF hand domain of PvCDPK4 was expressed in Echerichia coli and a 34 kDa product was obtained. Immunofluorescence assay by confocal laser microscopy revealed that the protein was expressed at the mature schizont of P. vivax. The expression of PvCDPK4-EF in schizont suggests that it may participate in the proliferation or egress process in the life cycle of this parasite.

• 대한수의학회지
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• 제37권3호
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• pp.541-546
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• 1997

VT2(Verotoxin-2)의 효소활성 영역에 해당되는 두 영역의 아미노산들에 대하여, 첫번째 보존영역의 Glu167을 conservative point mutation 시키고, 두 번째 보존영역의 구성 아미노산 5개 전부를 deletion mutation 시켜, 각 변이주에서 독성의 감소 정도를 wild type과 비교한 결과 다음과 같은 성적을 얻었다. 1. pKSC101을 Eco RI과 Pst I으로 절단하여 940bp insert를 통일 제한효소로 절단한 M 13mp19에 삽입하여 pEP19RF를 구축하였다. 이를 이용하여 dU-SSDNA template를 제조하고, mutagenic primer를 annealing 하여 변이를 도입하였으며, 변이가 도입된 insert를 acceptor plasmid에 삽입시켜 각각 발현 플라스미드 pOEX와 pDEX를 구축하였다. 각각의 mutant 단백질을 발현시키기 위하여 pOEX와 pDEX를 JM109에 형질전환시켜 mutant 재조합 균주인 POMUT109와 DEMUT109를 작성하였다. 2. POMUT109와 DEMUT109를 IPTG 유도 발현시킨 배양상층액을 Vero cell에 대하여 세포독성을 시험한 결과 wild type에 비하여 POMUT109의 배양 여액에서는 2000배, DEMUT109의 배양여액에서는 적어도 3000배 이상의 세포독성을 감소시켰다.