• Journal of Microbiology and Biotechnology
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• v.25 no.12
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• pp.2125-2134
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• 2015

IrrE is a highly conserved global regulator in the Deinococcus genus and contributes to survival from high doses of UV radiation, ionizing radiation, and desiccation. Drad-IrrE and Dgob-IrrE from Deinococcus radiodurans and Deinococcus gobiensis I-0 each share 66% sequence identity. However, Dgob-IrrE showed a stronger protection phenotype against UV radiation than Drad-IrrE in the D. radiodurans irrE-deletion mutant (ΔirrE), which may be due to amino acid residues differences around the DNA-binding HTH domain. Site-directed mutagenesis was used to generate a Drad-IrrE A184S single mutant, which has been characterized and compared with the ΔirrE mutant complemented strain with Drad-irrE, designated ΔirrE-E. The effects of the A184S mutation following UV radiation and mitomycin C (MMC) shock were determined. The A184S mutant displayed significantly increased resistance to UV radiation and MMC shock. The corresponding A184 site in Dgob-IrrE was inversely mutated, generating the S131A mutant, which exhibited a loss of resistance against UV radiation, MMC shock, and desiccation. qPCR analysis revealed that critical genes in the DNA repair system, such as recA, pprA, uvrA, and ddrB, were remarkably induced after UV radiation and MMC shock in the ΔirrE-IE and A184S mutants. These data suggested that A184S improves the ability against UV radiation and MMC shock, providing new insights into the modification of IrrE. We speculated that the serine residue may determine the efficiency of DNA binding, leading to the increased expression of IrrE-dependent genes important for protection against DNA damage.

• The Journal of Korean Society of Virology
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• v.30 no.1
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• pp.39-49
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• 2000

The genome of Hantaan virus, the prototype of the hantavirus genus, is composed of three segmented, single stranded negative sense RNA genome. The 5' and 3' termini of the Hantaan virus RNA genome contain noncoding regions (NCRs) that are highly conserved and complementary to form panhandle structures. There are some reports that these NCRs seems to control gene expression and viral replication in influenza virus and vesicular stomatitis virus. In this study, we examined whether NCRs in Hantaan virus playa role in expression of the viral nucleocapsid protein (Np) and foreign (luciferase) gene. The 5' and/or 3' NCR-deleted mutants were constructed and analysed. The Np expression of 5' NCR-deleted clone was similar to that of the clone containing full S genome. In the case of 3' NCR-deleted clone, it showed 40% reduction. To investigate the role of NCR in foreign gene expression, the clones which are replaced ORF of Hantaan viral Np gene with that of luciferase gene were constructed. The results were similar to those of the experiments using Np gene. These results suggest that 3' NCR is more important than 5' NCR in protein expression. To find out a critical region of 3' NCR in protein expression, several clones with a deleted part of 3' NCR were constructed and analyzed. The deletion of the conserved region in 3' NCR showed $20{\sim}30%$ decrease in Np expression. However there were no change in luciferase activities between clones with or without non-conserved region of 3' NCR. These results suggest that the 3' NCR of Hantaan virus S genome, especially conserved region in 3' NCR, plays an important role in the expression of Hantaan viral Np and foreign genes.

• Journal of Life Science
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• v.22 no.10
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• pp.1428-1433
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• 2012

Longevity is an exciting but difficult subject to study because it is determined by complex processes that require the coordinated action of several genetic factors as well as physiological and environmental influences. Genetic approaches have been applied to animal models to identify the molecular mechanism responsible for longevity. Several experimental model organisms obtained over the last decades suggest that the complete deletion of a single gene by gene targeting has proven to be an invaluable tool for the discovery of the mechanisms underlying longevity. The first discovery of long-lived mutants came from Caenorhabditis elegans research, which identified the insulin/IGF-1 pathway as responsible for longevity in this worm. IGF-1 is a multifunctional polypeptide that has sequence similarity to insulin and is involved in normal growth and development of cells. Several factors in the IGF-1 system have since been studied by gene targeting in the control of longevity in lower species, including nematode and fruit fly. In addition, significant progress has been made using mice models to extend the lifespan by targeted mutations that interfere with growth hormone/IGF-1 and IGF-1 signaling cascades. A recent finding that IGF-1 is involved in aging in mice was achieved by using liver-specific knockout mutant mice, and this clearly demonstrated that the IGF-1 signal pathway can extend the lifespan in both invertebrates and vertebrate models. Although the underlying molecular mechanisms for the control of longevity are not fully understood, it is widely accepted that reduced IGF-1 signaling plays an important role in the control of aging and longevity. Several genes involved in the IGF-1 signaling system are reviewed in relation to longevity in genetically modified mice models.

• Korean Journal of Microbiology
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• v.54 no.2
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• pp.91-97
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• 2018

The evolutionally conserved TREX complex member, Yra1/ALY, belongs to the REF (RNA and export factor binding proteins) family of hnRNP-like proteins, which has been implicated in multiple processes including transcription, nuclear RNA stability, and mRNA export. Fission yeast, Schizosaccharomyces pombe, genome encodes two members of REF proteins. In addition to Mlo3 known previously as an mRNA export factor, there is the other REF protein, Tho4, which is predicted as a component of THO complex. Here we showed that deletion of tho4 (SPBC106.12c) gene does not inhibit both growth and nuclear mRNA export. However, overexpression of tho4 displays growth retardation and slight accumulation of $poly(A)^+$ RNA in the nucleus. Neither ${\Delta}tho4$ ${\Delta}mlo3$ nor ${\Delta}tho4$ ${\Delta}mex67$ double mutants exhibit additive growth defect. Moreover, yeast two-hybrid and co-immunoprecipitation analysis did not show that the Tho4 protein interacted with any members of TREX complex and mRNA export factor Rae1. Contrary to expectation, these observations support that the S. pombe Tho4 is not a component of TREX complex, and not directly involved in bulk mRNA export from the nucleus.

• Journal of Life Science
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• v.17 no.2 s.82
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• pp.167-173
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• 2007

To study the transcriptional mechanisms by which expression of the murine glial cell-derived neurotrophic factor inducible transcription factor (mGIF) gene is regulated, a murine genomic clone was iso-lated using a mGIF cDNA as probe. A 13-kb genomic fragment, which comprises 4-kb upstream of the transcription initiation site was sequenced. The promoter region lacks a TATA box and CAAT box, is rich in G+C content, and has multiple putative binding sites for the transcription factor Spl. The mGIF gene also has consensus sequences for AP2 binding sites. The transcriptional activity of five deletion mutants of a 2.1-kb fragment was analyzed by modulating transcription of the heterologous luciferase gene in the promoterless plasmid pGL2-Basic. All mutants showed significant transcriptional activity in the murine neuroblastoma cell line NB41A3. Transient expression assays suggested the presence of a positive regulator between -213 and -129 while a negative regulator was found in the region between -806 and -214. Relatively strong transcriptional activity was observed in neuronal NB41A3, glial C6 cells and hepatic HepG2, but very weak activity in skeletal muscle C2C12 cells. These findings confirm the tissue-specific activity of the mGIF promoter and suggest that this gene shares structural and functional similarities with the dopamine receptor genes that it regulates.

• 한국균학회소식:학술대회논문집
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• 2015.05a
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• pp.59-59
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• 2015

Cryptococcus neoformans causes life-threatening meningoencephalitis in humans, but the treatment of cryptococcosis remains challenging. To develop novel therapeutic targets and approaches, signaling cascades controlling pathogenicity of C. neoformans have been extensively studied but the underlying biological regulatory circuits remain elusive, particularly due to the presence of an evolutionarily divergent set of transcription factors (TFs) in this basidiomycetous fungus. In this study, we constructed a high-quality of 322 signature-tagged gene deletion strains for 155 putative TF genes, which were previously predicted using the DNA-binding domain TF database (http://www.transcriptionfactor.org/). We tested in vivo and in vitro phenotypic traits under 32 distinct growth conditions using 322 TF gene deletion strains. At least one phenotypic trait was exhibited by 145 out of 155 TF mutants (93%) and approximately 85% of the TFs (132/155) have been functionally characterized for the first time in this study. Through high-coverage phenome analysis, we discovered myriad novel TFs that play critical roles in growth, differentiation, virulence-factor (melanin, capsule, and urease) formation, stress responses, antifungal drug resistance, and virulence. Large-scale virulence and infectivity assays in insect (Galleria mellonella) and mouse host models identified 34 novel TFs that are critical for pathogenicity. The genotypic and phenotypic data for each TF are available in the C. neoformans TF phenome database (http://tf.cryptococcus.org). In conclusion, our phenome-based functional analysis of the C. neoformans TF mutant library provides key insights into transcriptional networks of basidiomycetous fungi and ubiquitous human fungal pathogens.

• Journal of Life Science
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• v.26 no.9
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• pp.991-998
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• 2016

NF-κB acts as a critical transcription factor for the survival of cells via the induction of antiapoptotic genes. Constitutive activation of NF-κB in many types of solid tumors suggests that the inhibition of NF-κB might prevent or inhibit tumorigenesis. Although a number of studies demonstrated that Hsp70 regulated NF-κB activity, the exact mechanism is not clear. This study investigated the functional relationship of Hsp70 and IKKγ in the regulation of NF-κB activation using expression plasmids of components of the IKK complex. Wild-type and deletion mutants of IKKγ were expressed together with Hsp70, and the combined regulatory effect of Hsp70 and IKKγ on NF-κB activation was assayed. Hsp70 suppressed the activation of NF-κB in a reporter plasmid assay. Hsp70 also suppressed the phosphorylation and degradation of IκBα. The suppressive effect of Hsp70 on NF-κB activation was synergistically elevated by IKKγ. The N-terminal IKKβ binding site, C-terminal leucine zipper, and zinc finger domains of IKKγ were not necessary for the suppressive effect. Furthermore, Hsp70 and IKKγ synergistically suppressed the induction of COX-2 expression by lipopolysaccharides in RAW264.7 cells. These results suggest that overexpression of Hsp70 and IKKγ may be a strategic method for inhibition of NF-κB and related diseases.