• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2000년도 추계학술발표대회
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• pp.15-23
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• 2000

The subcellular localization of the SopB protein, which is encoded by the Escherichia coli F plasmid and is involved in the partition of the single-copy plasmid, was directly visualized through the expression of the protein fused to the jellyfish green fluorescent protein (GFP). The fusion protein was found to localize to positions close but not at the poles of exponentially growing cells. Examination of derivatives of the fusion protein lacking various regions of SopB suggests that the signal for the cellular localization of SopB resides in a region close to its N terminus. Overexpression of SopB led to silencing of genes linked to, but well-separated from, a cluster of SopB-binding sites termed sopC. In this SopB-mediated repression of sopC-linked genes, all but the N-terminal 82 amino acids of SopB can be replaced by the DNA-binding domain of a sequence-specific DNA -binding protein, provided that the sopC locus is also replaced by the recognition sequence of the DNA-binding domain. These results suggest a mechanism of gene silencing: patches of closely packed DNA-binding protein is localized to specific cellular sites; such a patch can capture a DNA carrying the recognition site of the DNA -binding domain and sequestrate genes adjacent to the recognition site through nonspecific binding of DNA.

• Parasites, Hosts and Diseases
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• 제61권4호
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• pp.397-404
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• 2023

Acanthamoeba species are free-living amoebae those are widely distributed in the environment. They feed on various microorganisms, including bacteria, fungi, and algae. Although majority of the microbes phagocytosed by Acanthamoeba spp. are digested, some pathogenic bacteria thrive within them. Here, we identified the roles of 3 phagocytosis-associated genes (ACA1_077100, ACA1_175060, and AFD36229.1) in A. castellanii. These 3 genes were upregulated after the ingestion of Escherichia coli. However, after the ingestion of Legionella pneumophila, the expression of these 3 genes was not altered after the consumption of L. pneumophila. Furthermore, A. castellanii transfected with small interfering RNS (siRNA) targeting the 3 phagocytosis-associated genes failed to digest phagocytized E. coli. Silencing of ACA1_077100 disabled phagosome formation in the E. coli-ingesting A. castellanii. Alternatively, silencing of ACA1_175060 enabled phagosome formation; however, phagolysosome formation was inhibited. Moreover, suppression of AFD36229.1 expression prevented E. coli digestion and consequently led to the rupturing of A. castellanii. Our results demonstrated that the ACA1_077100, ACA1_175060, and AFD36229.1 genes of Acanthamoeba played crucial roles not only in the formation of phagosome and phagolysosome but also in the digestion of E. coli.

• Plant Biotechnology Reports
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• 제4권3호
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• pp.185-192
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• 2010

The Arabidopsis ${\omega}$-3 fatty acid desaturase (AtFAD7) catalyzes the synthesis of trienoic fatty acids (TA). A transgenic tobacco line, T15, was produced by a sense AtFAD7 construct and showed a cosuppression-like phenotype, namely extremely low TA levels. The sequence similarity between AtFAD7 and a tobacco ortholog gene, NtFAD7, was moderate (about 69%) in the coding sequences. AtFAD7 siRNAs accumulated at a high level, and both AtFAD7 and NtFAD7 mRNAs are degraded in T15 plants. The low-TA phenotype in T15 was dependent on a tobacco RNA-dependent RNA polymerase6 (NtRDR6). We also produced tobacco RNAi plants targeting AtFAD7 gene sequences. The AtFAD7 siRNA level was trace, which was associated with a slight reduction in leaf TA level. Unexpectedly, this RNAi plant showed an increased NtFAD7 transcript level. To investigate the effect of translational inhibition on stability of the NtFAD7 mRNAs, leaves of the wild-type tobacco plants were treated with a translational inhibitor, cycloheximide. The level of NtFAD7 mRNAs significantly increased after cycloheximde treatment. These results suggest that the translational inhibition by low levels of AtFAD7 siRNAs or by cycloheximide increased stability of NtFAD7 mRNA. The degree of silencing by an RNAi construct targeting the AtFAD7 gene was increased by co-existence of the AtFAD7 transgene, where NtRDR6-dependent amplification of siRNAs occurred. These results indicate that NtRDR6 can emphasize silencing effects in both cosuppression and RNAi.

• Molecules and Cells
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• 제28권1호
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• pp.57-65
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• 2009

CDC48 is a member of the AAA ATPase superfamily. Yeast CDC48 and its mammalian homolog p97 are implicated in diverse cellular processes, including mitosis, membrane fusion, and ubiquitin-dependent protein degradation. However, the cellular functions of plant CDC48 proteins are largely unknown. In the present study, we performed virus-induced gene silencing (VIGS) screening and found that silencing of a gene encoding a tobacco CDC48 homolog, NgCDC48, resulted in severe abnormalities in leaf and shoot development in tobacco. Furthermore, transgenic tobacco plants (35S:anti-NgCDC48), in which the NgCDC48 gene was suppressed using the antisense RNA method, exhibited severely aberrant development of both vegetative and reproductive organs, resulting in arrested shoot and leaf growth and sterile flowers. Approximately 57-83% of 35S:anti-NgCDC48 plants failed to develop mature organs and died at early stage of development. Scanning electron microscopy showed that both adaxial and abaxial epidermal pavement cells in antisense transgenic leaves were significantly smaller and more numerous than those in wild type leaves. These results indicate that NgCDC48 is critically involved in cell growth and development of tobacco plants. An in vivo targeting experiment revealed that NgCDC48 resides in the endoplasmic reticulum (ER) in tobacco protoplasts. We consider the tantalizing possibility that CDC48-mediated degradation of an as-yet unidentified protein(s) in the ER might be a critical step for cell growth and expansion in tobacco leaves.

• BMB Reports
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• 제47권4호
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• pp.192-196
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• 2014

RNA polymerase II carboxyl-terminal domain (pol II CTD) phosphatases are a newly emerging family of phosphatases that are members of DXDX (T/V). The subfamily includes Small CTD phosphatases, like SCP1, SCP2, SCP3, TIMM50, HSPC129 and UBLCP. Extensive study of SCP1 has elicited the diversified roles of the small C terminal domain phosphatase. The SCP1 plays a vital role in various biological activities, like neuronal gene silencing and preferential Ser5 dephosphorylation, acts as a cardiac hypertrophy inducer with the help of its intronic miRNAs, and has shown a key role in cell cycle regulation. This short review offers an explanation of the mechanism of action of small CTD phosphatases, in different biological activities and metabolic processes.

• Molecules and Cells
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• 제20권2호
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• pp.167-172
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• 2005

Ceruloplasmin (Cp) is a copper protein with important functions in iron homeostasis and in inflammation. Cp mRNA expression is induced by interferon (IFN)-${\gamma}$ in U937 monocytic cells, but synthesis of Cp protein is halted after about 12 h by transcript-specific translational silencing. The silencing mechanism requires binding of a 4-component cytosolic inhibitor complex, IFN-gamma-activated inhibitor of translation (GAIT), to a defined structural element (GAIT element) in the Cp 3'-UTR. Translational silencing of Cp mRNA requires the essential proteins of mRNA circularization, suggesting that the translational inhibition requires end-to-end mRNA closure. These studies describe a new mechanism of translational control, and may shed light on the role that macrophage-derived Cp plays at the intersection of iron homeostasis and inflammation.

• Asian Pacific Journal of Cancer Prevention
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• 제16권7호
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• pp.3061-3065
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• 2015

Background: Rac3, a member of the Rac family of small guanosine triphosphatases (GTPases), regulates a variety of cell functions, including the organization of the cytoskeleton, cell migration, and invasion. Overexpression of Rac3 has been reported in several human cancers. However, the role of Rac3 in lung cancer (LC) has not been determined in detail. The purpose of this study was to investigate the effect of silencing of Rac3 expression in human LC cells and the consequences for cell survival. Materials and Methods: Lentivirus small hairpin RNA (shRNA) interference techniques were utilized to knock down the Rac3 gene. Gene and protein expression was quantified by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. LC cell apoptosis was examined by annexin V-APC /propidium iodide staining. Results: Efficient silencing of Rac3 strongly inhibited A549 cell proliferation and colony formation ability, and significantly decreased tumor growth. Moreover, flow cytometry analysis showed that knockdown of Rac3 led to G2/M phase cell cycle arrest as well as an excess accumulation of cells in the G1 and S phase. Conclusions: Thus, functional analysis using shRNAs revealed a critical role for Rac3 in the tumor growth of LC cells. shRNA silencing of Rac3 could provide an effective strategy to treat LC.

• Journal of Plant Biotechnology
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• 제37권2호
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• pp.115-124
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• 2010

As the completion of genome sequencing, large collection of expression data and the great efforts in annotating plant genomes, the next challenge is to systematically assign functions to all predicted genes in the genome. Functional genome analysis of plants has entered the high-throughput stage. The generations and collections of mutants at the genome-wide level form technological platform of functional genomics. However, to identify the exact function of unknown genes it is necessary to understand each gene's role in the complex orchestration of all gene activities in the plant cell. Gene function analysis therefore necessitates the analysis of temporal and spatial gene expression patterns. The most conclusive information about changes in gene expression levels can be gained from analysis of the varying qualitative and quantitative changes of messenger RNAs, proteins and metabolites. New technologies have been developed to allow fast and highly parallel measurements of these constituents of the cell that make up gene activity. We have reviewed currently employed technologies to identify unknown functions of predicted genes including map-based cloning, insertional mutagenesis, reverse genetics, chemical mutagenesis, microarray analysis, FOX-hunting system, gene silencing mutagenesis, proteomics and chemical genomics. Recent improvements in technologies for functional genomics enable whole-genome functional analysis, and thus open new avenues for studies of the regulations and functions of unknown genes in plants.

• 한국고분자학회지:고분자과학과기술
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• 제23권3호
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• pp.254-259
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• 2012

• 한국동물학회:학술대회논문집
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• 한국동물학회 2000년도 한국생물과학협회 학술발표대회
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• pp.59.2-59
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• 2000

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