• 생명과학회지
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• 제16권3호
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• pp.454-458
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• 2006

출아효모인 Sacharomyces cerevisiae S288C균주를 이용한 효모의 게놈이 완성된 후 S. cerevisiae는 다양한 연구 모델로 이용되어져 왔다. 현재까지 효모를 이용한 기능 유전체학 측면에서의 연구는 laboratory strainin인 S288C 균주 또는 그 유래의 균주들이다. 그러나 자연에서 분리된 효모 또는 산업적으로 이용되어지고 있는 S. cerevisiae의 유전학 측면에서의 연구는 낮은 포자형성률 및 형질전환률, 그리고 S288C 균주와의 게놈상의 상이성 때문에 거의 이루어지지 않고 있다. 여기서 우리 연구진은 자연에서 분리된 Saccharomyces cerevisiae KNU5377 균주를 이용하여 random spore analysis를 통해 MATa 및 $MAT{\alpha}$ 타입의 각각의 haploid cell을 분리 후 이미 보고된 KanMX module를 가지고 round PCR기법에 의한 short flanking homology 기법을 이용하여 전사조절인자인 HSF1 유전자가 치환된 변이주를 구축할 수 있었다. 덧붙여, 모든 유전자에 이 기법을 적용할 수는 없다는 것을 확인하였다. 앞으로 이 변이주를 통해 기능 유전체학적인 측면에서 이 유전자의 스트레스와의 관련성을 연구하고자 한다.

• Journal of Plant Biotechnology
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• 제1권1호
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• pp.13-19
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• 1999

The visco-elastic properties of gluten are major determinants of the processing properties of doughs. These visco-elastic properties are strongly influenced by the ratio of monomeric and polymeric proteins and the size distribution of the polymeric proteins, which make up the gluten fraction of the dough. Recent studies have revealed that other features, such as the number of the cysteine residues of the HMW-GS, also play an important role in determining the functional characteristics. To modify the processing properties at molecular level, the relationship between the structure of molecules and dough properties has to be understood. In order to explore the relationships between individual proteins and dough properties, we have developed procedures for incorporating bacterially expressed proteins into doughs, and measuring their functional properties in small-scale equipment. A major problem in investigating the structure/function relationships of individual seed storage proteins is to obtain sufficient amounts of pure polypeptides from the complex families of proteins expressed in the endosperm. Therefore, we have established a simplified model system in which we produce specific protein genes through bacterial expression and test their functional properties in smallscale apparatus after incorporation into base flour. An S poor protein gene has been chosen as a template gene. This template gene has been modified using standard recombinant DNA techniques in order to test the effects of varying the number and position of cysteine residues, and the size of the protein. Doughs have been mixed in small scale apparatus and characterized with respect to their polymeric composition and their functional properties, including dough mixing, extensibility and small scale bating. We conclude that dough characteristics can be manipulated in a predictable manner by altering the cysteine residues and the size of high molecular weight glutenins.

• Mycobiology
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• 제49권5호
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• pp.491-497
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• 2021

An endolichenic fungus Xylaria grammica EL000614 produces grammicin, a potent nematicidal pyrone derivative that can serve as a new control option for root-knot nematodes. We optimized an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for X. grammica to support genetic studies. Transformants were successfully generated after co-cultivation of homogenized young mycelia of X. grammica with A. tumefaciens strain AGL-1 carrying a binary vector that contains the bacterial hygromycin B phosphotransferase (hph) gene and the eGFP gene in T-DNA. The resulting transformants were mitotically stable, and PCR analysis showed the integratin of both genes in the genome of transformants. Expression of eGFP was confirmed via fluorescence microscopy. Southern analysis showed that 131 (78.9%) out of 166 transformants contained a single T-DNA insertion. Crucial factors for producing predominantly single T-DNA transformants include 48 h of co-cultivation, pretreatment of A. tumefaciens cells with acetosyringone before co-cultivation, and using freshly prepared mycelia. The established ATMT protocol offers an efficient tool for random insertional mutagenesis and gene transfer in studying the biology and ecology of X. grammica.

• Biotechnology and Bioprocess Engineering:BBE
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• 제7권5호
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• pp.237-251
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• 2002

The recent progress on metabolic systems engineering was reviewed based on our recent research results in terms of (1) metabolic signal flow diagram approach, (2) metabolic flux analysis (MFA) in particular with intracellular isotopomer distribution using NMR and/or GC-MS, (3) synthesis and optimization of metabolic flux distribution (MFD), (4) modification of MFD by gene manipulation and by controlling culture environment, (5) metabolic control analysis (MCA), (6) design of metabolic regulation structure, and (7) identification of unknown pathways with isotope tracing by NMR. The main characteristics of metabolic engineering is to treat metabolism as a network or entirety instead of individual reactions. The applications were made for poly-3-hydroxybutyrate (PHB) production using Ralstonia eutropha and recombinant Escherichia coli, lactate production by recombinant Saccharomyces cerevisiae, pyruvate production by vitamin auxotrophic yeast Toluropsis glabrata, lysine production using Corynebacterium glutamicum, and energetic analysis of photosynthesic microorganisms such as Cyanobateria. The characteristics of each approach were reviewed with their applications. The approach based on isotope labeling experiments gives reliable and quantitative results for metabolic flux analysis. It should be recognized that the next stage should be toward the investigation of metabolic flux analysis with gene and protein expressions to uncover the metabolic regulation in relation to genetic modification and/ or the change in the culture condition.

• Journal of Animal Science and Technology
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• 제55권4호
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• pp.303-314
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• 2013

Knowledge regarding lipid catabolism has been of great interest in the field of animal sciences. In the livestock industry, excess fat accretion in meat is costly to the producer and undesirable to the consumer. However, intramuscular fat (marbling) is desirable to enhance carcass and product quality. The manipulation of lipid content to meet the goals of animal production requires an understanding of the detailed mechanisms of lipid catabolism to help meticulously design nutritional, pharmacological, and physiological approaches to regulate fat accretion. The concept of a basic system of lipases and their co-regulators has been identified. The major lipases cleave triacylglycerol (TAG) stored in lipid droplets in a sequential manner. In adipose tissue, adipose triglyceride lipase (ATGL) performs the first and rate-limiting step of TAG breakdown through hydrolysis at the sn-1 position of TAG to release a non-esterified fatty acid (NEFA) and diacylglycerol (DAG). Subsequently, cleavage of DAG occurs via the rate-limiting enzyme hormone-sensitive lipase (HSL) for DAG catabolism, which is followed by monoglyceride lipase (MGL) for monoacylglycerol (MAG) hydrolysis. Recent identification of the co-activator (Comparative Gene Identification-58) and inhibitor [G(0)/G(1) Switch Gene 2] of ATGL have helped elucidate this important initial step of TAG breakdown, while also generating more questions. Additionally, the roles of these lipolysis-related enzymes in muscle, liver and skin tissue have also been found to be of great importance for the investigation of systemic lipolytic regulation.

• Journal of Plant Biotechnology
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• 제39권1호
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• pp.1-12
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• 2012

With the increasing advances in Brassicaceae genetics and genomics, considerable progress has been made in the transformation of Brassicaceae. Transformation technologies are now being exploited routinely to determine the gene function and contribute to the development of novel enhanced crops. $Agrobacterium$-mediated transformation remains the most widely used approach for the introduction of transgenes into Brassicaceae. In $Brassica$, the transformation relies mainly on $in$ $vitro$ transformation methods. Nevertheless, despite the significant progress made towards enhancing the transformation efficiencies, some genotypes remain recalcitrant to transformation. Advances in our understanding of the genetics behind various transformations have enabled researchers to identify more readily transformable genotypes for use in routine high-throughput systems. These developments have opened up exciting new avenues to exploit model $Brassica$ genotypes as resources for understanding the gene function in complex genomes. Although many other Brassicaceae have served as model species for improving plant transformation systems, this paper summarizes on the recent technologies employed in the transformation of both $Arabidopsis$ and $Brassica$. The use of transformation technologies for the introduction of desirable traits and a comparative analysis of these as well as their future prospects are also important parts of the current research that is reviewed.

• Asian-Australasian Journal of Animal Sciences
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• 제16권6호
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• pp.910-927
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• 2003

Transgenesis is a very powerful tool not only to help understanding the basics of life science but also to improve the efficiency of animal production. Since the first transgenic mouse was born in 1980, rapid development and wide application of this technique have been made in laboratory animals as well as in domestic animals. Although pronuclear injection is the most widely used method and nuclear transfer using somatic cells broadens the choice of making transgenic domestic animals, the demand for precise manipulation of the genome leads to the utilization of gene targeting. To make this technique possible, a pluripotent embryonic cell line such as embryonic stem (ES) cell is required to carry genetic mutation to further generations. However, ES cell, well established in mice, is not available in domestic animals even though many attempt to establish the cell line. An alternate source of pluripotent cells is embryonic germ (EG) cells derived from primordial germ cells (PGCs). To make gene targeting feasible in this cell line, a better culture system would help to minimize the unnecessary loss of cells in vitro. In this review, general methods to produce transgenic domestic animals will be mentioned. Also, it will focus on germ cell engineering and methods to improve the establishment of pluripotent embryonic cell lines in domestic animals.

• 한국미생물·생명공학회지
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• 제23권2호
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• pp.164-169
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• 1995

An endogenous cryptic plasmid, pBL1, which has been used to construct plasmid vectors for coryneform bacteria producing amino acids, was eliminated from Brevibacterium lactofermentum. The pBL1 was partially digested with Sau3AI and the resulting DNA fragments were subcloned into a suicide vector pEM1 which contains a kanamycin-resistant (km$^{r}$) gene. KM$^{r}$ B. lactofermentum transconjugants were obtained by conjugal transfer of the pEM1 derivatives containing pBL1 DNA fragments from Escherichia coli into B. lactofermentum. A km$^{r}$ transconjugant was analyzed to contain a plasmid pEB14, which occurred in vivo by homologous recombination between pBL1 and the conjugal-transferred plasmid. The pEB14 including the pEM1-derived km$^{r}$ gene was found to be lost concomitantly with km$^{r}$ phenotype, resulting in the construction of a pBL1-free strain of B lactofermentum. Based on transformation efficiencies and plasmid stability, the resultant pBL1- free strain is more useful than wild strain as a host cell for genetic manipulation. It could be concluded that foreign plasmid DNAs are efficiently isolated and analyzed from the pBL1-free strain because of the absence of endogenous pBL1 plasmid.

• Mycobiology
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• 제49권6호
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• pp.599-603
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• 2021

CRISPR/Cas9 genome editing systems have been established in a broad range of eukaryotic species. Herein, we report the first method for genetic engineering in pyogo (shiitake) mushrooms (Lentinula edodes) using CRISPR/Cas9. For in vivo expression of guide RNAs (gRNAs) targeting the mating-type gene HD1 (LeA1), we identified an endogenous LeU6 promoter in the L. edodes genome. We constructed a plasmid containing the LeU6 and glyceraldehyde-3-phosphate dehydrogenase (LeGPD) promoters to express the Cas9 protein. Among the eight gRNAs we tested, three successfully disrupted the LeA1 locus. Although the CRISPR-Cas9-induced alleles did not affect mating with compatible monokaryotic strains, disruption of the transcription levels of the downstream genes of LeHD1 and LeHD2 was detected. Based on this result, we present the first report of a simple and powerful genetic manipulation tool using the CRISPR/Cas9 toolbox for the scientifically and industrially important edible mushroom, L. edodes.

• KSBB Journal
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• 제20권4호
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• pp.323-327
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• 2005

조직을 이용한 역전사 (RT)-PCR법을 이용하면 원하는 특정유전자의 발현을 비교적 정확하게 알 수 있지만 조직의 RNA를 이용하므로 세포단위의 정확한 유전자 발현을 알기에는 한계가 있다. 특히 그 기능과 성질이 다른 세포가 무수하게 많이 혼재하는 두뇌와 같은 조직은 신경계의 각종 뉴런(신경세포), 글리어 (glial cell) 등이 서로 얽혀 있다. 대표적인 신경세포의 degeneration 질병으로는 파킨슨병 (Parkinson's disease; PD)이 있다. 파킨슨병은 사람의 신경세포 관련 질병에 있어서 가장 일반적인 질병의 하나이다. PD의 가장 중요한 원인은 도파민 생성 신경세포의 퇴행 혹은 사멸에 기인하여 도파민 (dopamine)이라는 신경전달물질이 감소하는 것이 그 원인이다. 도파민과 같은 카테콜아민의 생합성에 관련된 효소는 타이로신 하이드록실레이스 (TH), 도파 데카르복실레이스 (DDC) 등이 알려져 있다. 그러나 그런 효소들의 생화학적 연구는 많이 되어 있음에도 불구하고 단일 흑질 신경세포에서의 이들 관련 유전자의 발현 양상에 대해서는 알려진 바가 거의 없다. PD와 관련된 유전자의 발현 정도를 밝히기 위하여, 레이저 다이섹터 (laser micro-dissector)에 의한 단일 신경세포의 분리에 착수하였다. 정해진 방법에 따라 정상 대조구 (비PD)와 PD 환자에서 각각 한 개 또는 여러 개를 성공적으로 분리한 흑질 신경세포를 이용하여 유전자 특이적 프라이머를 사용하여 RT-PCR을 행하였다. 그 결과, 단 한 개의 신경세포에서도 여러 개의 세포를 사용한 것과 같은 동일한 결과를 얻는 데 성공하였다. PD환자의 뇌에서 분리한 10개의 독립적인 세포의 예에서는 각 세포간의 발현차이가 인정되었으며, 특히 TH 유전자의 발현은 상당히 높은 확률로 검출되지 않았다. 이 결과로 단일 신경세포에서의 mRNA양을 검출하기 위해서는 본 본문의 RT-PCR법이 효과적인 방법임을 알 수 있다.