• Mycobiology
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• 제43권1호
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• pp.31-36
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• 2015

We have previously isolated ${\varepsilon}$-COP, the ${\alpha}$-COP interactor in COPI of Aspergillus nidulans, by yeast two-hybrid screening. To understand the function of ${\varepsilon}$-COP, the $aneA^+$ gene for ${\varepsilon}$-COP/AneA was deleted by homologous recombination using a gene-specific disruption cassette. Deletion of the ${\varepsilon}$-COP gene showed no detectable changes in vegetative growth or asexual development, but resulted in decrease in the production of the fruiting body, cleistothecium, under conditions favorable for sexual development. Unlike in the budding yeast Saccharomyces cerevisiae, in A. nidulans, over-expression of ${\varepsilon}$-COP did not rescue the thermo-sensitive growth defect of the ${\alpha}$-COP mutant at $42^{\circ}C$. Together, these data show that ${\varepsilon}$-COP is not essential for viability, but it plays a role in fruiting body formation in A. nidulans.

• 미생물학회지
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• 제46권4호
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• pp.401-404
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• 2010

출아효모 Saccharomyces cerevisiae에서 RNA-binding 단백질인 Tho1은 mRNA가 전사되는 동안 초기 mRNA에 결합하여 mRNP 생성과 성숙한 mRNA의 핵에서 세포질로의 방출에 관여하는 것으로 여겨진다. 분열효모 Schizosaccharomyces pombe에서도 Tho1과 유사한 단백질을 암호화하는 유전자(spTho1로 명명)를 찾아 그 특성을 조사하였다. 이배체 S.pombe 균주에 하나의 spTho1 유전자만을 결실시킨 후 4분체분석을 수행한 결과, 이 유전자는 생장에 반드시 필요하지 않았다. 또한 spTho1 결실 돌연변이는 mRNA의 핵에서 세포질로의 방출도 정상적으로 보였다. 그러나 티아민에 의해 발현이 조절되는 강력한 프로모터를 이용하여 spTho1를 과발현시키면, 세포의 생장이 억제되었으며 $poly(A)^+$ RNA가 핵 안에 축적되었다. 이와 같은 결과들은 spTho1 유전자가 필수적이지는 않지만 mRNA의 핵에서 세포질로의 방출에 관여하고 있음을 시사한다.

• 미생물학회지
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• 제44권1호
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• pp.80-84
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• 2008

mRNA의 핵에서 세포질로의 이동에 중요한 역할을 하는 발아효모 Saccharomyces cerevisiae의 DEAD-box RNA helicase인 DBP5 유전자와 유사한 분열효모 Schizosaccharomyces pombe의 유전자(spDbp5로 명명)의 결실돌연변이주(knockout mutant)를 제조하여 그 특성을 조사하였다. 이배체인 S. pombe 균주에 하나의 spDbp5 유전자만을 결실시킨 후 4분체분석(tetrad analysis)을 수행한 결과, 이 유전자가 결실된 반수체 균주는 생장하지 못했다. mRNA의 핵에서 세포질로의 이동에 있어서 spDbp5의 역할을 알아보기 위해, spDbp5의 발현이 티아민(thiamin)에 의해 억제되는 균주를 제작하여 in situ hybridization을 통해 세포 내의 $poly(A)^+$ RNA 분포를 살펴보았다. spDbp5 유전자의 발현이 억제되면, $poly(A)^+$ RNA가 핵 안에 축적되고세포질에서는 줄어들었다. 이와 같은 결과들은 spDbp5 유전자 역시 mRNA의 핵에서 세포질로의 이동에 매우 중요한 역할을 담당하고 있음을 시사한다.

• Journal of Microbiology and Biotechnology
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• 제17권9호
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• pp.1504-1512
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• 2007

The fpr gene, which encodes a ferredoxin-$NADP^+$ reductase, is known to participate in the reversible redox reactions between $NADP^+$/NADPH and electron carriers, such as ferredoxin or flavodoxin. The role of Fpr and its regulatory protein, FinR, in Pseudomonas putida KT2440 on the oxidative and osmotic stress responses has already been characterized [Lee at al. (2006). Biochem. Biophys. Res. Commun. 339, 1246-1254]. In the genome of P. putida KT2440, another Fpr homolog (FprB) has a 35.3% amino acid identity with Fpr. The fprB gene was cloned and expressed in Escherichia coli. The diaphorase activity assay was conducted using purified FprB to identify the function of FprB. In contrast to the fpr gene, the induction of fprB was not affected by oxidative stress agents, such as paraquat, menadione, $H_2O_2$, and t-butyl hydroperoxide. However, a higher level of fprB induction was observed under osmotic stress. Targeted disruption of fprB by homologous recombination resulted in a growth defect under high osmotic conditions. Recovery of oxidatively damaged aconitase activity was faster for the fprB mutant than for the fpr mutant, yet still slower than that for the wild type. Therefore, these data suggest that the catalytic function of FprB may have evolved to augment the function of Fpr in P. putida KT2440.

• Reproductive and Developmental Biology
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• 제35권4호
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• pp.511-518
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• 2011

To avoid hyperacute rejection of xenografts, ${\alpha}1,3$-galactosyltransferase knock-out (GalT KO) pigs have been produced. In this study, we examined whether Sia-containing glycoconjugates are important as an immunogenic non-Gal epitope in the pig liver with disruption of ${\alpha}1,3$-galactosyltransferase gene. The target cells were then used as donor cells for somatic cell nuclear transfer (scNT). A total of 1,800 scNT embryos were transferred to 10 recipients. One recipient developed to term and naturally delivered two piglets. Real-time RT-PCR and glycosyltransferase activity showed that ${\alpha}2,3$-sialyltransferase (${\alpha}2,3ST$) and ${\alpha}2,6$-sialyltransferase (${\alpha}2,6ST$) in the heterozygote GalT KO liver have higher expression levels and activities compared to controls, respectively. According to lectin blotting, sialic acidcontaining glycoconjugate epitopes were also increased due to the decreasing of ${\alpha}$-Gal in heterozygote GalT KO liver, whereas GalNAc-containing glycoconjugate epitopes were decreased in heterozygote GalT KO liver compare to the control. Furthermore, the heterozygote GalT KO liver showed a higher Neu5Gc content than control. Taken together, these finding suggested that the deficiency of GalT gene in pigs resulted in increased production of Neu5Gc-bounded epitopes (H-D antigen) due to increase of ${\alpha}2,6$-sialyltransferase. Thus, this finding suggested that the deletion of CMAH gene to the GalT KO background is expected to further prolong xenograft survival.

• Journal of Microbiology and Biotechnology
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• 제27권2호
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• pp.335-341
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• 2017

The complexity of the bacterial recombination system is a barrier for the construction of bacterial mutants for the further functional investigation of specific genes. Several protocols have been developed to inactivate genes from the genus Pseudomonas. Those protocols are complicated and time-consuming and mostly do not enable easy construction of multiple knock-ins/outs. The current study describes a single and double crossover-recombination system using an optimized vector-free allele-exchange protocol for gene disruption and gene replacement in a single species of the family Pseudomonadaceae. The protocol is based on self-ligation (circularization) for the DNA cassette which has been obtained by overlapping polymerase chain reaction (Fusion-PCR), and carries an antibiotic resistance cassette flanked by homologous internal regions of the target locus. To establish the reproducibility of the approach, three different chromosomal genes (ncRNA31, rpoN, rpoS) were knocked-out from the root-associative bacterium Pseudomonas stutzeri A1501. The results showed that the P. stutzeri A1501 mutants, which are free of any plasmid backbone, could be obtained via a single or double crossover recombination. In order to optimize this protocol, three key factors that were found to have great effect on the efficiency of the homologous recombination were further investigated. Moreover, the modified protocol does not require further cloning steps, and it enables the construction of multiple gene knock-in/out mutants sequentially. This work provides a simple and rapid mutagenesis strategy for genome editing in P. stutzeri, which may also be applicable for other gram-negative bacteria.

• 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.

• 미생물학회지
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• 제45권4호
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• pp.300-305
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• 2009

mRNA의 3' 말단형성 뿐만 아니라, 성숙한 mRNA의 핵에서 세포질로의 이동에 중요한 역할을 하는 출아효모 Saccharomyces cerevisiae의 폴리(A)-RNA 결합단백질인 Nab2와 유사한 분열효모 Schizosaccharomyces pombe의 단백질을 암호화하는 유전자(spNab2로 명명)의 결실돌연변이주(deletion mutant)를 제조하여 그 특성을 조사하였다. 이배체인 S. pombe 균주에 하나의 spNab2 유전자만을 결실시킨 후 4분체분석(tetrad analysis)을 수행한 결과, S. cerevisiae NAB2와는 다르게 이 유전자는 생장에 반드시 필요하지 않았다. 또한 spNab2 결실돌연변이는 mRNA의 핵에서 세포질로의 이동도 정상적으로 보였다. spNab2의 역할을 알아보기 위해, 티아민에 의해 발현이 조절되는 강력한 프러모터를 이용하여 spNab2를 과발현시켰다. spNab2 유전자가 과발현되면, 세포의 생장이 심하게 억제되었으며, 폴리(A)-RNA가 핵 안에 축적되고 세포질에서는 줄어들었다. 또한 GFP 융합단백질을 이용하여 spNab2 단백질의 세포 내 위치를 관찰한 결과, spNab2-GFP는 주로 핵 안에 존재하였지만 세포질에서도 관찰되었다. 이와 같은 결과들은 spNab2 유전자 역시 mRNA의 핵에서 세포질로의 이동에 관여하고 있음을 시사한다.

• Laboraroty Animal Research
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• 제34권4호
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• pp.257-263
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• 2018

Trefoil factor 1 (TFF1, also known as pS2) is strongly expressed in the gastrointestinal mucosa and plays a critical role in the differentiation of gastric glands. Since approximately 50% of all human gastric cancers are associated with decreased TFF1 expression, it is considered a tumor suppressor gene. Tff1 deficiency in mice results in histological changes in the antral and pyloric gastric mucosa, with severe hyperplasia and dysplasia of epithelial cells, resulting in the development of antropyloric adenoma. Here, we generated Tff1-knockout (KO) mice, without a neomycin resistant ($Neo^R$) cassette, using the clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRSIPR/Cas9) system. Though our Tff1-KO mice showed phenotypes very similar to the previous embryonic stem (ES)-cell-based KO mice, they differed from the previous reports in that a reduction in body weight was observed in males. These results demonstrate that these newly established Tff1-KO mice are useful tools for investigating genetic and environmental factors influencing gastric cancer, without the effects of artificial gene insertion. Furthermore, these findings suggest a novel hypothesis that Tff1 expression influences gender differences.

• Genomics & Informatics
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• 제19권4호
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• pp.47.1-47.12
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• 2021

Kaposi's sarcoma-associated herpesvirus (KSHV) is one of the few human oncogenic viruses, which causes a variety of malignancies, including Kaposi's sarcoma, multicentric Castleman disease, and primary effusion lymphoma, particularly in human immunodeficiency virus patients. The currently available treatment options cannot always prevent the invasion and dissemination of this virus. In recent times, siRNA-based therapeutics are gaining prominence over conventional medications as siRNA can be designed to target almost any gene of interest. The ORF57 is a crucial regulatory protein for lytic gene expression of KSHV. Disruption of this gene translation will inevitably inhibit the replication of the virus in the host cell. Therefore, the ORF57 of KSHV could be a potential target for designing siRNA-based therapeutics. Considering both sequence preferences and target site accessibility, several online tools (i-SCORE Designer, Sfold web server) had been utilized to predict the siRNA guide strand against the ORF57. Subsequently, off-target filtration (BLAST), conservancy test (fuzznuc), and thermodynamics analysis (RNAcofold, RNAalifold, and RNA Structure web server) were also performed to select the most suitable siRNA sequences. Finally, two siRNAs were identified that passed all of the filtration phases and fulfilled the thermodynamic criteria. We hope that the siRNAs predicted in this study would be helpful for the development of new effective therapeutics against KSHV.