• BMB Reports
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• v.28 no.4
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• pp.331-335
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• 1995

Simian virus 40 (SV40) small-t antigen (small-t) has been known to regulate the activity of a cellular enzyme, protein phosphatase 2A (PP2A), composed of A. B, and C subunits, via binding to the A subunit In the study presented here, the stoichiometry of the binding of small-t to PP2A was determined to be 1: 1. It was also shown that small-t binds to the AC form of PP2A with a higher apparent affinity than it binds to the free A subunit. We also characterized the interaction of PP2A with wild-type and various mutant small-ts. A single-point mutant (Val134Met) and a double-point mutant (Trp147Gly;Leu152 Pro) of small-t exhibited 3-fold and 5-fold lower potencies in inhibiting PP2A activity. respectively. This suggests that the region around amino acids between 134 and 152 of small-t might be important in regulating the enzyme activity of PP2A.

• Microbiology and Biotechnology Letters
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• v.25 no.4
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• pp.379-385
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• 1997

Several protease-deficient mutants of Aspergillus niger have been isolated by halo-screening on skim milk plate after UV irradiation of conidiospores. The extracellular proteolytic activities of the mutant strains grown in an optimized medium varied from 3% to 85% of that of the parental strain. Especially, two mutant strains named as ANPD-129 and ANPD-153, which had 3% and 49% of acid protease activity of the parental strain, respectively, were further characterized both physiologically and genetically. The growth rates of the mutants, ANPD-129 and ANPD-153, were similar to that of the parental strain, unlike other protease-deficient mutants. The diploid formed between the two mutants restored protease activity to a similar level of that of the parental strain. This result revealed that ANPD-129 and ANPD-153 had mutations at different loci. Using master strains with marked chromosomes these loci were assigned to linkage groups. The mutation locus (prt129) in ANPD-129 was assigned to linkage group VI and the locus (prt153) in ANPD-153 to linkage group III.

• Journal of Microbiology and Biotechnology
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• v.17 no.5
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• pp.721-727
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• 2007

Stability-enhanced mutants, H44, 11-94, 5A2-84, and F8, of L-threonine aldolase(L-TA) from Streptomyces coelicolor A3(2)(SCO1085) were isolated by an error-prone PCR followed by a high-throughput screening. Each of these mutant, had a single amino acid substitution: H177Y in the H44 mutant, A169T in the 11-94 mutant, D104N in the 5A2-84 mutant and F18I in the F8 mutant. The residual L-TA activity of the wild-type L-TA after a heat treatment for 20 min at $60^{\circ}C$ was only 10.6%. However, those in the stability-enhanced mutants were 85.7% for the H44 mutant, 58.6% for the F8 mutant, 62.1% for the 5A2-84 mutant, and 67.6% for the 11-94 mutant. Although the half-life of the wild-type L-TA at $63^{\circ}C$ was 1.3 min, those of the mutant L-TAs were longer: 14.6 min for the H44 mutant, 3.7 min for the 11-94 mutant, 5.8 min for the 5A2-84 mutant, and 5.0 min for the F8 mutant. The specific activity did not change in most of the mutants, but it was decreased by 45% in the case of mutant F8. When the aldol condensation of glycine and 3,4-dihydroxybenzaldehyde was studied by using whole cells of Escherichia coli containing the wild-type L-TA gene, L-threo-3,4-dihydroxyphenylserine(L-threo-DOPS) was successfully synthesized with a yield of 2.0 mg/ml after 20 repeated batch reactions for 100 h. However, the L-threo-DOPS synthesizing activity of the enzyme decreased with increased cycles of the batch reactions. Compared with the wild-type L-TA, H44 L-TA kept its L-threo-DOPS synthesizing activity almost constant during the 20 repeated batch reactions for 100 h, yielding 4.0 mg/ml of L-threo-DOPS. This result showed that H44 L-TA is more effective than the wild-type L-TA for the mass production of L-threo-DOPS.

• Journal of Microbiology and Biotechnology
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• v.20 no.4
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• pp.718-726
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• 2010

A selected fungal strain, for production of the raw-starchdigesting enzyme by solid-state fermentation, was improved by two repeated sequential exposures to ${\gamma}$-irradiation of $Co^{60}$, ultraviolet, and four repeated treatments with Nmethyl-N'-nitrosoguanidine. The mutant strain Aspergillus sp. XN15 was chosen after a rigorous screening process, with its production of the raw-starch-digesting enzyme being twice that of usual wild varieties cultured under preoptimized conditions and in an unsupplemented medium. After 17 successive subculturings, the enzyme production of the mutant was stable. Optimal conditions for the production of the enzyme by solid-state fermentation, using wheat bran as the substrate, were accomplished for the mutant Aspergillus sp. XN15. With the optimal fermentation conditions, and a solid medium supplemented with nitrogen sources of 1% urea and 1% $NH_4NO_3$, 2.5 mM $CoSO_4$, 0.05% (v/w) Tween 80, and 1% glucose, the mutant Aspergillus sp. XN15 produced the raw-starch-digesting enzyme in quantities 19.4 times greater than a typical wild variety. Finally, XN15, through simultaneous saccharification and fermentation of a raw rice corn starch slurry, produced a high level of ethanol with $Y_{p/s}$ of 0.47 g/g.

• The Plant Pathology Journal
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• v.24 no.1
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• pp.1-7
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• 2008

Fusarium verticillioides (teleomorph Gibberella moniliformis) is associated with maize worldwide causing ear rot and stalk rot, and produces fumonisins, a group of mycotoxins detrimental to humans and animals. While research tools are available, our understanding of the molecular mechanisms associated with fungal virulence and fumonisin biosynthesis in F. verticillioides is still limited. One of the restraints that hampers F. verticillioides gene characterization is the fact that homologous recombination (HR) frequency is very low (<2%). Screening for a true gene knock-out mutant is a laborious process due to a high number of ectopic integrations. In this study, we generated a F. verticillioides mutant (SF41) deleted for FvKU70, a gene directly responsible for non-homologous end-joining mechanism, with the aim of improving HR frequency. Here, we demonstrate that FvKU70 deletion does not impact key Fverticillioides phenotypes, e.g., development, secondary metabolism, and virulence, while dramatically improving HR frequency. Significantly, we also confirmed that a high percentage (>85%) of the HR mutant strains harbor a desired mutation with no additional copy of the mutant allele inserted in the genome. We conclude that SF41 is suitable for use as a type strain when performing high-throughput gene function studies in F. verticillioides.

• Journal of Microbiology and Biotechnology
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• v.15 no.5
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• pp.1022-1027
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• 2005

Based on plasmid display technology by the complexes of fusion protein and the encoding plasmid DNA, an in vitro selection method for high affinity DNA-binding protein was developed and experimentally demonstrated. The GAL4 DNA-binding domain (GAL4 DBD) was selected as a model DNA-binding protein, and enhanced green fluorescent protein (EGFP) was used as an expression reporter for the selection of target proteins. Error prone PCR was conducted to construct a mutant library of the model. Based on the affinity decrease with increased salt concentration, mutants of GAL4 DBD having high affinity were selected from the mutant protein library of protein-encoding plasmid complex by this method. Two mutants of (Lys33Glu, Arg123Lys, Ile127Lys) and (Ser47Pro, Ser85Pro) having high affinity were obtained from the first generation mutants. This method can be used for rapid in vitro selection of high affinity DNA-binding proteins, and has high potential for the screening of high affinity DNA-binding proteins in a sequence-specific manner.

• Biomolecules & Therapeutics
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• v.31 no.1
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• pp.48-58
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• 2023

Interferon regulatory factor 3 (IRF3) integrates both immunological and non-immunological inputs to control cell survival and death. Small GTPases are versatile functional switches that lie on the very upstream in signal transduction pathways, of which duration of activation is very transient. The large number of homologous proteins and the requirement for site-directed mutagenesis have hindered attempts to investigate the link between small GTPases and IRF3. Here, we constructed a constitutively active mutant expression library for small GTPase expression using Gibson assembly cloning. Small-scale screening identified multiple GTPases capable of promoting IRF3 phosphorylation. Intriguingly, 27 of 152 GTPases, including ARF1, RHEB, RHEBL1, and RAN, were found to increase IRF3 phosphorylation. Unbiased screening enabled us to investigate the sequence-activity relationship between the GTPases and IRF3. We found that the regulation of IRF3 by small GTPases was dependent on TBK1. Our work reveals the significant contribution of GTPases in IRF3 signaling and the potential role of IRF3 in GTPase function, providing a novel therapeutic approach against diseases with GTPase overexpression or active mutations, such as cancer.

• The Korean Journal of Oral and Maxillofacial Pathology
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• v.42 no.6
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• pp.159-165
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• 2018

The Hippo pathway was originally discovered in Drosophila by genetic screening and it has been shown to be conserved in various organisms including human. Until now, the essential roles of Hippo pathway in regulating cell proliferation, apoptosis, tumorigenesis, and organ size control is extensively studied. Currently, Mats1/2 (Mob1a/1b), one of the important components in Hippo pathway, mutant mice were generated which has abnormal phenotype such as resistance to apoptosis and spontaneous tumorigenesis. Of note, Mats1/2 mutant mice also showed dental malocclusion. Therefore, in this study, we have evaluated the bone phenotype of Mats1/2 mutant mice. Although the mRNA expressions of Mats1 or Mats2 were observed in both osteoclastogenesis and osteoblastogenesis, the increase of Mats1 level was most prominent during osteoblastogenesis. The RANKL-induced osteoclast differentiation from bone marrow-derived macrophages (BMMs) was unaltered upon Mats1/2 mutation; however, the osteoblast differentiation using calvarial pre-osteoblasts was significantly reduced in Mats1/2 mutant mice compare to that of wild type mice. In accordance with in vitro results, Mats1/2 mutant mice showed decreased bone volume as well as increased trabecular separation in ${\mu}CT$ analyses. These results may provide novel prospect of the probable linkage between Hippo pathway and bone homeostasis.

• Plant Biotechnology Reports
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• v.5 no.1
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• pp.45-51
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• 2011

A low acid phosphatase 3 (lap3) mutant was identified and characterized from an Arabidopsis activation-tagged (Weigel) population. The roots of the lap3 plants showed lower acid phosphatase (APase) activity compared to wild-type ones under low-Pi conditions ($10{\mu}M\;Pi$). Plasmid rescue experiments revealed that the activation-tagging vector was inserted into the intergenic region between At4g31540 and At4g31550 in the Arabidopsis genome. The genotypic segregation of the lap3 mutation was tightly linked with the phenotypic segregation of root APase activity in the prgeny of lap3. The transcript level of the At4g31520 (SDA1: SEVERE DEPOLYMERIZATION OF ACTIN 1), located 7.4 kb from the CaMV 35S enhancers in the lap3 mutant, was significantly reduced compared to that in the wild type. It was speculated that cellular actin polymerization may be involved in Pi acquisition in higher plants.

• Journal of Life Science
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• v.17 no.5 s.85
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• pp.641-645
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• 2007

We conducted a seed germination screening under saline conditions to identify salt tolerance(sto) mutants with ethyl methane sulfonate(EMS) mutagenesis seed pool. During the screening, we identified three mutant lines that seemed to confer elevated salt tolerance in high concentrations of NaCl. At 175 mM NaCl, germination rate of sto42-14 mutant(one of the EMS salt tolerance mutants) was 7-fold higher than that of wild-type plants. Interestingly, sto42-14 mutant exhibited insensitivity to high glucose concentration and growth inhibition to gibberellin. Our results suggest that sto42-14 is involved in salt stress tolerance as well as in glucose and gibberellin response in Arabidopsis.