• Molecules and Cells
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• 제38권10호
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• pp.866-875
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• 2015

COPI vesicles are essential to the retrograde transport of proteins in the early secretory pathway. The COPI coatomer complex consists of seven subunits, termed ${\alpha}-$, ${\beta}-$, ${\beta}^{\prime}-$, ${\gamma}-$, ${\delta}-$, ${\varepsilon}-$, and ${\zeta}$-COP, in yeast and mammals. Plant genomes have homologs of these subunits, but the essentiality of their cellular functions has hampered the functional characterization of the subunit genes in plants. Here we have employed virus-induced gene silencing (VIGS) and dexamethasone (DEX)-inducible RNAi of the COPI subunit genes to study the in vivo functions of the COPI coatomer complex in plants. The ${\beta}^{\prime}-$, ${\gamma}-$, and ${\delta}$-COP subunits localized to the Golgi as GFP-fusion proteins and interacted with each other in the Golgi. Silencing of ${\beta}^{\prime}-$, ${\gamma}-$, and ${\delta}$-COP by VIGS resulted in growth arrest and acute plant death in Nicotiana benthamiana, with the affected leaf cells exhibiting morphological markers of programmed cell death. Depletion of the COPI subunits resulted in disruption of the Golgi structure and accumulation of autolysosome-like structures in earlier stages of gene silencing. In tobacco BY-2 cells, DEX-inducible RNAi of ${\beta}^{\prime}$-COP caused aberrant cell plate formation during cytokinesis. Collectively, these results suggest that COPI vesicles are essential to plant growth and survival by maintaining the Golgi apparatus and modulating cell plate formation.

• Journal of Microbiology
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• 제37권4호
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• pp.248-255
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• 1999

We have analyzed the MRE3/REC114 gene of Saccharomyces cerevisiae, previously detected in isolation of mutants defective in meiotic recombination. We cloned the MRE3/REC114 gene by complementation of the meiotic recombination defect and it has been mapped to chormosome XIII. The DNA sequence analysis revealed that the MRE3 gene is identical to the REC114 gene. The upstream region of the MRE3/REC114 gene contains a T_4C site, a URS (upstream repression sequence) and a TR (T-rich) box-like sequence, which reside upstream of many meiotic genes. Coincidentally, northern blot analysis indicated that the three sizes of MRE3/REC114 transcripts, 3.4, 1.4 and 1.2 kb, are induced in meiosis. A less abundant transcript of 1.4 kb is detected in both mitotic and meiotic cells, suggesting that it is needed in mitosis as well as meiosis. To examine the role of the MRE3/REC114 gene, we constructed mre3 disruption mutants. Strains carrying an insertion or null deletion of the MRE3/REC114 gene showed slow growth in nutrient medium and the doubling time of these cells increased approximately by 2-fond compared to the wild-type strain. Moreover, the deletion mutant (${\delta}$mre3) displayed no meiotically induced recombination and no viable spores. The mre3/rec114 spore lethality can be suppressed by spo13, a mutation that causes cells to bypass reductional division. The double-stranded breaks (DSBs) which are involved in initiation of meiotic recombination were not detected in the analysis of meiotic chromosomal DNA from the mre3/rec114 disruptant. From these results we suggest that the MRE3/REC114 gene product is essential in normal growth and in early meiotic stages involved in meiotic recombination.

• International Journal of Industrial Entomology and Biomaterials
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• 제37권1호
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• pp.15-20
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• 2018

Most proteins produced in the endoplasmic reticulum (ER) of eukaryotic cells fold via disulfide formation (oxidative folding). Oxidative folding is catalyzed by protein disulfide isomerase (PDI) and PDI-related ER protein thiol disulfide oxidoreductases (ER oxidoreductases). In yeast and mammals, ER oxidoreductin-1s (ERO1s) supply oxidizing equivalent to the active centers of PDI. We previously identified and characterized the ERO1 of Bombyx mori (bERO1) as a thioredoxin-like protein that shares primary sequence homology with other ERO1s. Here we compare the reactivation of inactivated rRNase and sRNase by bERO1, and show that bERO1 and bPDI cooperatively refold denatured RNase A. This is the first result suggesting that bERO1 plays an essential role in ER quality control through the combined activities of bERO1 and bPDI as a catalyst of protein folding in the ER and sustaining cellular redox homeostasis.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
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• pp.380-383
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• 2000

불완전 곰팡이의 일종인 Aureobasidium pullulans ATCC 42023 균주를 이용한 pullulan의 최적조건 및 pH 조절에 따른 pullulan의 생성 그리고 균체형태와 고분자 pullulan 생산에 미치는 영향을 조사 하였다. 삼각플라스크 내에서 최대의 pullulan 생산은 초기 pH 6.5일때 11.98 g/l의 pullulan을 생산하였고, 회분식 발효에서는 산소 공급속도 0.5 vvm일때 15.16 g/l의 최대의 pullulan생산량을 나타내었다. pH조절에 따른 실험에서는 pH 4.5일때 13.31 g/l의 pullulan 생산량을 보였고, 이때 균체의 형태는 효모형과 균사형태의 성장을 하였다. 그러나 분자량 생성면에서는 pH 6.5일때 100만 이상의 고분자가 가장 많이 생산되었다.

• Journal of Microbiology and Biotechnology
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• 제20권12호
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• pp.1664-1671
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• 2010

A new biosurfactant producer was isolated from palm-oil-contaminated soil and later identified through morphology and DNA sequencing as the yeast-like fungus Exophiala dermatitidis. Biosurfactant production was catalyzed by vegetable oil, supplemented with a basal medium. The culture conditions that provided the biosurfactant with the highest surface activity were found to be 5% palm oil with 0.08% $NH_4NO_3$, at a pH of 5.3, with shaking at 200 rpm, and a temperature of $30^{\circ}C$ for a 14-day period of incubation. The biosurfactant was purified, in accordance with surfactant properties, by solvent fractionation using silica gel column chromatography. The chemical structure of the strongest surface-active compound was elucidated through the use of NMR and mass spectroscopy, and noted to be monoolein, which then went on to demonstrate antiproliferative activity against cervical cancer (HeLa) and leukemia (U937) cell lines in a dose-dependent manner. Interestingly, no cytotoxicity was observed with normal cells even when high concentrations were used. Cell and DNA morphological changes, in both cancer cell lines, were observed to be cell shrinkage, membrane blebbling, and DNA fragmentation.

• Mycobiology
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• 제45권4호
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• pp.434-437
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• 2017

An improved method for extracting high quality and quantity RNA from a jelly mushroom and a dimorphic fungus-Tremella fuciformis which is especially rich in polysaccharides, is described. RNA was extracted from T. fuciformis mycelium M1332 and its parental monokaryotic yeast-like cells Y13 and Y32. The A260/280 and A260/230 ratios were both approximately 2, and the RNA integrity number was larger than 8.9. The yields of RNA were between 108 and $213{\mu}g/g$ fresh wt. Downstream molecular applications including reverse transcriptional PCR and quantitative real-time PCR were also performed. This protocol is reliable and may be widely applicable for total RNA extraction from other jelly mushrooms or filamentous fungi rich in polysaccharides.

• Molecules and Cells
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• 제45권3호
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• pp.158-167
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• 2022

Ubiquitin (Ub) is post-translationally modified by Ub itself or Ub-like proteins, phosphorylation, and acetylation, among others, which elicits a variety of Ub topologies and cellular functions. However, N-terminal (Nt) modifications of Ub remain unknown, except the linear head-to-tail ubiquitylation via Nt-Met. Here, using the yeast Saccharomyces cerevisiae and an Nt-arginylated Ub-specific antibody, we found that the detectable level of Ub undergoes Nt-Met excision, Nt-deamination, and Nt-arginylation. The resulting Nt-arginylated Ub and its conjugated proteins are upregulated in the stationary-growth phase or by oxidative stress. We further proved the existence of Nt-arginylated Ub in vivo and identified Nt-arginylated Ub-protein conjugates using stable isotope labeling by amino acids in cell culture (SILAC)-based tandem mass spectrometry. In silico structural modeling of Nt-arginylated Ub predicted that Nt-Arg flexibly protrudes from the surface of the Ub, thereby most likely providing a docking site for the factors that recognize it. Collectively, these results reveal unprecedented Nt-arginylated Ub and the pathway by which it is produced, which greatly expands the known complexity of the Ub code.

• 농업과학연구
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• 제15권1호
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• pp.15-22
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• 1988

팽이버섯 원형질체(原形質體)의 재생(再生)과 환원(還元)에 영향(影響)을 미치는 요인(要因)에 대해 조사(調査)하였으며 영양요구성(營養要求性) 돌연변이(突然變異) 균주(菌株) 선발(選拔)을 실시(實施)하여 얻은 결과(結果)는 다음과 같다. 1. 원형질체(原形質體) 재생배지(再生培地)에 첨가(添加)되는 삼투압 조절제(調節劑)로는 0.6M의 sucrose가 가장 우수(優秀)하였고 재생률(再生率)은 0.47~1.32%로 나타났다. 재생용(再生用) 배지(培地)에 몇가지의 영양원(營養源)을 첨가(添加)하였을때 재생률(再生率)의 증가(增加)를 나타내었으며 특히 yeast extract가 팽이버섯의 원형질체(原形質體) 재생(再生)에 효과적(效果的)이었다. 2. 팽이버섯의 원형질체(原形質體) 재생(再生) 형태(形態)는 bud-like cells에서 germ tube가 발달(發達)하는 형태(形態)이었다. 환원(還元)된 균총중(菌叢中)에는 13~18%의 clamp connection을 형성(形成)하지 않는 monokaryon 균주(菌株)들이 발견(發見)되었다. 3. 자외선(U.V.L) 조사시(照射時) 팽이버섯의 포자생존률(胞子生存率)은 180사(砂)에서 16%였고 이때 5계통(系統)의 영양요구성(營養要求性) 균주(菌株)가 선발(選拔)되었다. 이들 중(中) 2균주(菌株)는 nucleic acid requiring strain이었고 3균주(菌株)는 vitamine requiring strain이었다.

• BMB Reports
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• 제51권11호
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• pp.578-583
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• 2018

Telomeres are specialized nucleoprotein complexes that function to protect eukaryotic chromosomes from recombination and erosion. Several telomere binding proteins (TBPs) have been characterized in higher plants, but their detailed in vivo functions at the plant level are largely unknown. In this study, we identified and characterized OsTRFL1 (Oryza sativa Telomere Repeat-binding Factor Like 1) in rice, a monocot model crop. Although OsTRFL1 did not directly bind to telomere repeats $(TTTAGGG){_4}$ in vitro, it was associated with telomeric sequences in planta. OsTRFL1 interacted with rice TBPs, such as OsTRBF1 and RTBP1, in yeast and plant cells as well as in vitro. Thus, it seems likely that the association of OsTRFL1 with other TBPs enables OsTRFL1 to bind to telomeres indirectly. T-DNA inserted OsTRFL1 knock-out mutant rice plants displayed significantly longer telomeres (6-25 kb) than those (5-12 kb) in wild-type plants, indicating that OsTRFL1 is a negative factor for telomere lengthening. The reduced levels of OsTRFL1 caused serious developmental defects in both vegetative and reproductive organs of rice plants. These results suggest that OsTRFL1 is an essential factor for the proper maintenance of telomeres and normal development of rice.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2017년도 9th Asian Crop Science Association conference
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• pp.110-110
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• 2017

Stomata are the main gateways for water and air transport between leaves and the environment. Inward-rectifying potassium channels regulate photo-induced stomatal opening. Rice contains three inward rectifying shaker-like potassium channel proteins, OsKAT1, OsKAT2 and OsKAT3. Among these, only OsKAT2 is specifically expressed in guard cells. Here, we investigated the functions of OsKAT2 in stomatal regulation using three dominant negative mutant proteins, OsKAT2(T235R), OsKAT2(T285A) and OsKAT2(T285D), which are altered in amino acids in the channel pore and at a phosphorylation site. Yeast complementation and patch clamp assays showed that all three mutant proteins lost channel activity. However, among plants overexpressing these mutant proteins, only plants overexpressing OsKAT2(T235R) showed significantly less water loss than the control. Moreover, overexpression of this mutant protein led to delayed photo-induced stomatal opening and increased drought tolerance. Our results indicate that OsKAT2 is an inward-rectifying shaker-like potassium channel that mainly functions in stomatal opening. Interestingly, overexpression of OsKAT2(T235R) did not cause serious defects in growth or yield in rice, suggesting that OsKAT2 is a potential target for engineering plants with improved drought tolerance without yield penalty.