• 한국식물학회:학술대회논문집
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• 한국식물학회 1996년도 제10회 식물생명공학심포지움 고등식물 발생생물학의 최근 진보
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• pp.61-74
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• 1996

Human and monogastric animals can not synthesize 10 out of the 20 amino asids and therefor need to obtain these from their diet. The plant seed is a major source of dietary protein. It is particular important in their study to increase nutritional quality of the seed storage proteins. The low contents of lysine, asparagine and threonenein various cereal seeds and of cystein and methionine. In legume seeds is due to the low proportions of these amino acids in the major storage proteins, we have tried to apply the three strategies; (1) mutagenesis and selection of specific amino acid analogue resistance, (2) cloning and expression study of lysine biosynthesis related gene, (3) transfomation of lysine rich soybean glycinin gene. The 5-methyltryptophan (5MT) resistant cell lines, SAR1, SAR2 and SAR3 were selected from anther derived callus of rice (Oryza sativa L. "Sasanishiki"). Among these selected cell lines, two (SAR1 and SAR3) were able to grow stably at 200 mg/L of 5MT. Analysis of the freed amino acids in callus shows that 5MT resistant cells (SAR3) accumulated free tryptophan at least up to 50 times higher than those that of the higher than of SAS. These results indicated that the 5MT resistant cell lines are useful in studies of amino acid biosynthesis. Tr75, a rice (Oryza sativa L., var. Sasanishiki) mutant resistant to 5MT was segregated from the progenies of its initial mutant line, TR1. The 5MT resistant of TR75 was inherited in the M8 generations as a single dominant nuclear gene. The content of free amino acids in the TR75 homozygous seeds increased approximately 1.5 to 2.0 fold compared to wild-type seeds. Especially, the contents of tryptophan, phenylalanine and aspartic acid were 5.0, 5.3 and 2.7 times higher than those of wild-type seeds, respectively. The content of lysine is significantly low in rice. The lysine is synthesized by a complex pathway that is predominantly regulated by feedback inhibition of several enzymes including asparginase, aspatate kinase, dihydrodipicolinat synthase, etc. For understanding the regulation mechanism of lysine synthesis in rice, we try to clone the lysine biosynthetic metabolism related gene, DHPS and asparaginase, from rice. We have isolated a rice DHPS genomic clone which contains an ORF of 1044 nucleotides (347 amino acids, Mr. 38, 381 daltons), an intron of 587 nucleotides and 5'and 3'-flanking regions by screening of rice genomic DNA library. Deduced amino acid sequence of mature peptide domain of GDHPS clone is highly conserved in monocot and dicot plants whereas that of transit peptide domain is extremely different depending on plant specie. Southern blot analysis indicated that GDHPS is located two copy gene in rice genome. The transcripts of a rice GDHPS were expressed in leaves and roots but not detected in callus tissues. The transcription level of GDHPS is much higher in leaves indicating enormous chloroplast development than roots. Genomic DNA clones for asparaginase genes were screened from the rice genomic library by using plaque hybridization technique. Twelve different genomic clones were isolated from first and second screening, and 8 of 12 clones were analyzed by restriction patterns and identified by Southern Blotting, Restriction enzyme digestion patterns and Southern blot analysis of 8 clones show the different pattern for asparaginase gene. Genomic Southern blot analysis from rice were done. It is estimated that rice has at least 2-3 copy of asparaginase gene. One of 8 positive clones was subcloned into the pBluescript SK(+) vector, and was constructed the physical map. For transformation of lysine rich storage protein into tobacco, soybean glycinin genes are transformed into tobacco. To examine whether glycinin could be stably accumulated in endosperm tissue, the glycinin cDNA was transcriptionally fused to an endosperm-specific promotor of the rice storage protein glutelin gene and then introduced into tobacco genomic via Agrobacterium-mediated transformation. Consequently the glycinin gene was expressed in a seed-and developmentally-specific manner in transgenic tobacco seeds. Glycinin were targeted to vacuole-derived protein bodies in the endosperm tissue and highly accumulated in the matrix region of many transgenic plant (1-4% of total seed proteins). Synthesized glycinin was processed into mature form, and assembled into a hexamer in a similar manner as the glycinin in soybean seed. Modified glycinin, in which 4 contiguous methionine residues were inserted at the variable regions corresponding to the C - teminal regions of the acidic and basic polypeptides, were also found to be accumulated similarly as in the normal glycinin. There was no apparent difference in the expression level, processing and targeting to protein bodies, or accumulation level between normal and modified glycinin. glycinin.

• 생명과학회지
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• 제9권3호
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• pp.253-261
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• 1999

Leptin gene, an obesity gene, has been known to involve in the regulation of food intake and body weight. It is also thought to be related to the glucose metabolism, insulin secretion and type II diabetes mellitus. Recently, the production of recombinant leptin protein has been attempted for the application in the treatment of obesity and the correction of hereditary obesity and type II diabetes. In the present study, leptin cDNA was cloned from mouse fat cells by RT-PCR and prokaryotic expression of leptin was attempted in order ot prepare a leptin-specific antigen. Immunization of a rabbit with the leptin-specific antigen into a rabbit resulted in the generation of leptin-specific antiserum that could be useful in the detection of leption expressed in various tissues. The sequence of leptin cDNA prepared in the present study wa identical to the previously reported one. Transformation of E. coli(DH5a) cells with the leptin cDNA-inserted translation vector, pGEX-4T-3-leptin followed by treatment with IPTG (0.1mM) resulted in the expression of a large amount of GST-leptin fusion protein with a molecular weight of 44 KDa as an inclusion body. Denaturation of the insoluble fusion protein by 8M urea, 6M guanidium-HCI or 0.1% 2-mercaptoethanol followed by a slow oxidation could not solubilize the inclusion body. The cell extract was subjected to SDS-PAGE and GST-leptin protein electroeluted from the gel was then injected into a rabbit subcutaneously for the immunization. Anti-GST-leptin rabbit antiserum which had a cross reactivity to the GST-leptin protein was generated. Leptin protein expressed in mouse brain and fat tissues was detected by Western blot immunodetection system using the antiserum generated in the present study.

• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2002년도 제38회 학술심포지움
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• pp.13-19
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• 2002

To elucidate the regulatory mechanism for expression of sialyl-glycoconjugates and their biological functions, ninetheen sialyltransferase cDNAs including eleven by our group or co-works have been cloned and characterized so far. The cloned sialyltransferases are classified into four families according to the carbohydrate linkages they synthesize: ${\alpha}2,3-sialyltransferase$ (ST3Gal I-VI), ${\alpha}$ 2,6-sialyltransferase (ST6Gal I), GalNAc ${\alpha}$ 2,6-sialyltransferase (ST6GalNAc I-VI), and ${\alpha}2,8-sialyltransferase$ (ST8Sia I-VI). Each of the sialyltransferase genes is differentially expressed in a tissue-, cell type-, and stage-specific manner. These enzymes differ in their substrate specificity and various biochemical parameters. However, enzymatic analysis conducted in vitro with recombinant enzyme revealed that one linkage can be synthesized by multiple enzymes. We present here an overview of structure and function of sialyltransferases performed by our group and co-works. Genomic structures and transcriptional regulation of two kinds of human sialyltransferase gene are also presented.

• International Journal of Oral Biology
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• 제32권1호
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• pp.35-43
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• 2007

Tooth loss in elderly is mainly caused by alveolar bone loss via severe periodontitis. Although the severity of periodontitis is known to be affected by age, the aging process or the genetic changes during the aging of periodontal tissue cells are not well characterized. In this study, we investigated the effect of in vitro aging on the change of gene expression pattern in periodontal fibroblasts. Gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDL) were obtained from two young patients and replicative senescence was induced by sequential subcultivation. When more than 90% cells were positively stained with senescence-associated ${\beta},-galactosidase$, those cells were regarded as aged cells. In aged GF and PDL, the level of phosphorylated retinoblastoma (RB) and $p16^{INK4A}$ protein was significantly decreased and increased, respectively. However, the protein level of p53 and p21, well known senescence-inducing genes, did not increase in aged GF and PDL. Although $p27^{Kip1}$ and $p15^{INK4B}$, another cyclin-dependent kinase inhibitors, were reported to be involved in replicative senescence of human cells, they were decreased in aged GF and PDL. Because senescent cells showed flattened and enlarged cell shape and are known to have increased focal adhesion, we examined the protein level of several integrins. Aged GF and PDL showed increased protein level of integrin ${\alpha}2$, ${\alpha}v$, and ${\beta}1$. When the gene expression profiles of actively proliferating young cells and aged cells were compared by cDNA microarray of 3,063 genes and were confirmed by reverse transcription-polymerase chain reaction, 7 genes and 15 genes were significantly and commonly increased and decreased, respectively, in aged GF and PDL. Among them, included are the genes that were known to be involved in the regulation of cell cycle, gene transcription, or integrin signaling. The change of gene expression pattern in GF and PDL was minimally similar to that of oral keratinocyte. These results suggest that $p16^{INK4A}/RB$ might be involved in replicative senescence of periodontal fibroblasts and the change of gene expression profile during aging process is cell type specific.

• BMB Reports
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• 제53권11호
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• pp.551-564
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• 2020

Proper development of the nervous system is critical for its function, and deficits in neural development have been implicated in many brain disorders. A precise and predictable developmental schedule requires highly coordinated gene expression programs that orchestrate the dynamics of the developing brain. Especially, recent discoveries have been showing that various mRNA chemical modifications can affect RNA metabolism including decay, transport, splicing, and translation in cell type- and tissue-specific manner, leading to the emergence of the field of epitranscriptomics. Moreover, accumulating evidences showed that certain types of RNA modifications are predominantly found in the developing brain and their dysregulation disrupts not only the developmental processes, but also neuronal activities, suggesting that epitranscriptomic mechanisms play critical post-transcriptional regulatory roles in development of the brain and etiology of brain disorders. Here, we review recent advances in our understanding of molecular regulation on transcriptome plasticity by RNA modifications in neurodevelopment and how alterations in these RNA regulatory programs lead to human brain disorders.

• 생명과학회지
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• 제30권8호
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• pp.713-721
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• 2020

Adipogenesis의 연구는 인간의 지방생물학의 기초적인 분자기전을 이해하고, 비만, 당뇨 및 대사성 증후군의 발병기전을 밝히는데 필요하다. Adipogenesis의 많은 연구가 adipocytes 특이적인 핵심 전사인자인 PPARγ와 C/EBPα를 중심으로 하는 유전자 발현조절 및 세포 내 신호전달에 초점이 맞추어 활발하게 연구가 진행되었다. 그러나, 에피지놈 변형효소나 히스톤 돌연변이에 의한 에피지놈 관점에서 adipogenesis 연구는 미흡한 실정이다. 포유동물에서 히스톤 methylation은 유전자 발현에 대한 주요 후성유전적(epigenome) 변형 중 하나이며, 특히 히스톤 H3 lysine methylation은 다양한 조직 및 기관 발생과정과 세포 분화에 매우 중요한 히스톤 변형이다. 세포 특이적 enhancer는 adipogenesis에서 active enhancer 표지자인 H3K27ac와 함께 H3K4me1로 변형된다. MLL4는 Pparg 및 Cebpa 유전자 ehancers에서 중요한 adipogenic H3K4 mono-methyltransferase이다. 따라서 MLL4는 adipogenesis에 중요한 에피지놈 변형효소라고 할 수 있다. 유전자 발현 억제를 유발하는 대표적인 히스톤 변형인 H3K27me3은 Polycomb repressive complex 2의 효소활성 subunit인 Ezh2에 의해 매개된다. Wnt 유전자에서 Ezh2에 의한 H3K27me3 히스톤 methylation 변형은 adipogenesis를 증가시키는데, 이는 WNT 신호 전달이 adipogenesis의 억제 조절자로 알려져 있기 때문이다. 본 논문은 유전자 발현을 근본적으로 조절하는 히스톤 H3 methylation에 의한 후성 유전학적인 조절이 어떻게 adipogenesis를 조절하는지에 대해 요약한다.

• Molecules and Cells
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• 제42권4호
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• pp.363-375
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• 2019

Fungal sectorization is a complex trait that is still not fully understood. The unique phenotypic changes in sporadic sectorization in mutants of CpBck1, a mitogen-activated protein kinase kinase kinase (MAPKKK) gene, and CpSlt2, a mitogen-activated protein kinase (MAPK) gene, in the cell wall integrity pathway of the chestnut blight fungus Cryphonectria parasitica have been previously studied. Although several environmental and physiological factors cause this sectoring phenotype, genetic variants can also impact this complex morphogenesis. Therefore, RNA sequencing analysis was employed to identify candidate genes associated with sectorization traits and understand the genetic mechanism of this phenotype. Transcriptomic analysis of CpBck1 and CpSlt2 mutants and their sectored progeny strains revealed a number of differentially expressed genes (DEGs) related to various cellular processes. Approximately 70% of DEGs were common between the wild-type and each of CpBck1 and CpSlt2 mutants, indicating that CpBck1 and CpSlt2 are components of the same MAPK pathway, but each component governs specific sets of genes. Functional description of the DEGs between the parental mutants and their sectored progenies revealed several key pathways, including the biosynthesis of secondary metabolites, translation, amino acid metabolism, and carbohydrate metabolism; among these, pathways for secondary metabolism and translation appeared to be the most common pathway. The results of this comparative study provide a better understanding of the genetic regulation of sector formation and suggest that complex several regulatory pathways result in interplays between secondary metabolites and morphogenesis.

• 생명과학회지
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• 제21권2호
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• pp.227-234
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• 2011

Pseudomonas syringae pv. tabaci는 숙주인 담배에 감염하여 들불병(wild fire)을 일으키는 식물 병원성 세균이다. 이 세균의 pathogenicity island (PAI)는 Type III secretion system 및 병원성 유전자들을 암호화하고 있으며, 병원성 조절에 있어 핵심적인 역할을 한다. 최근 식물 병원성 세균인 Ralstonia solanacearum에서 식물 세포 접촉을 매개로 하여 hrp gene cluster를 양성조절하는 PrhA (plant regulator of hrp) receptor가 발견되었다. 본 연구에서는 P. syringae에서 식물세포에 의해 hrp 유전자가 유도되는지 확인하기 위해, prhA 유사체를 동정하고 PrhA 결실돌연변이주(BL11)를 구축하였다. BL11은 숙주 감염 실험에서 병원성이 현저히 감소하였고, 식물 세포현탁액에서 hrpA 유전자의 발현수준이 hrp 유도배지에서 보다 3배 더 높게 나타났다. 이러한 결과들을 근거로 PrhA가 식물세포접촉에 의한 조절에 중요한 역할을 한다는 것을 확인하였으며, hrpA-gfp reporter fusion을 사용하여 이를 다시 검증하였다.

• IMMUNE NETWORK
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• 제14권3호
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• pp.138-148
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• 2014

MicroRNAs (miRNAs) are endogenous small RNA molecules best known for their function in post-transcriptional gene regulation. Immunologically, miRNA regulates the differentiation and function of immune cells and its malfunction contributes to the development of various autoimmune diseases including systemic lupus erythematosus (SLE). Over the last decade, accumulating researches provide evidence for the connection between dysregulated miRNA network and autoimmunity. Interruption of miRNA biogenesis machinery contributes to the abnormal T and B cell development and particularly a reduced suppressive function of regulatory T cells, leading to systemic autoimmune diseases. Additionally, multiple factors under autoimmune conditions interfere with miRNA generation via key miRNA processing enzymes, thus further skewing the miRNA expression profile. Indeed, several independent miRNA profiling studies reported significant differences between SLE patients and healthy controls. Despite the lack of a consistent expression pattern on individual dysregulated miRNAs in SLE among these studies, the aberrant expression of distinct groups of miRNAs causes overlapping functional outcomes including perturbed type I interferon signalling cascade, DNA hypomethylation and hyperactivation of T and B cells. The impact of specific miRNA-mediated regulation on function of major immune cells in lupus is also discussed. Although research on the clinical application of miRNAs is still immature, through an integrated approach with advances in next generation sequencing, novel tools in bioinformatics database analysis and new in vitro and in vivo models for functional evaluation, the diagnostic and therapeutic potentials of miRNAs may bring to fruition in the future.

• Asian-Australasian Journal of Animal Sciences
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• 제21권2호
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• pp.167-176
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• 2008

Galectins are a group of animal lectins consisting of galectin-type carbohydrate recognition domains (CRD) with relatively minor domains. The biological properties of galectins include the regulation of inflammation, intercellular adhesion, cell differentiation and cell death. The diverse kinds of galectin suggest variety in their biological roles. Galectin-1 is released during adipocyte differentiation and is associated with fat which is one of the important factors for meat quality. To verify expression level, a 0.5 kb clone of galectin-1 was obtained from cDNA prepared from back fat tissue of a Sancheong Berkshire pig with good quality meat, and the galectin-1 gene identified. The deduced amino acid sequence of the galectin-1 gene was compared with those obtained from other species. By using RT-PCR and Real time-PCR, an attempt was made to determine the expression level of galectin-1 and to compare with various tissues (tenderloin and back fat) taken from pigs in different groups. Grouping of pigs was based on growth-stage (weighing 60, 80, and 110 kg) and the sub-speciation (Yorkshire and Sancheong Berkshire pigs). We attempted to determine influences of pig species, growth stages and tissue variations on the expression level of the galectin-l gene and it was revealed that the expression pattern of the galectin-1 gene was significantly different (p<0.01 or p<0.05). Galectin-1 genes were expressed more highly in the back fat tissues of pigs weighing 110 kg than in those weighing 60 kg or 80 kg. However, the lowest expression was seen in the tenderloin tissues of pigs weighing 110 kg. Sancheong Berkshire pigs showed higher expression of the galectin-1 gene compared to Yorkshire pigs. Accordingly, it is considered that the expression pattern of the galectin-1 gene influences the growth of back fat tissues and the pig speciation relationship. Previous studies suggested that different expression of galectin-1 genes represents variety among the breeds and is closely related to fat tissue growth, conjugation and catabolism. Further, this study suggests that the expression of galectin-1 at a specific growth stage and tissue contributes significantly to the overall meat quality of Sancheong Berkshire pigs.