• Asian-Australasian Journal of Animal Sciences
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• v.29 no.10
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• pp.1383-1391
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• 2016

Bovine embryonic stem cells have potential for use in research, such as transgenic cattle generation and the study of developmental gene regulation. The Nanog may play a critical role in maintenance of the undifferentiated state of embryonic stem cells in the bovine, as in murine and human. Nevertheless, efforts to study the bovine Nanog for pluripotency-maintaining factors have been insufficient. In this study, in order to understand the mechanisms of transcriptional regulation of the bovine Nanog, the 5'-flanking region of the Nanog was isolated from ear cells of Hanwoo. Results of transient transfection using a luciferase reporter gene under the control of serially deleted 5'-flanking sequences revealed that the -134 to -19 region contained the positive regulatory sequences for the transcription of the bovine Nanog. Results from mutagenesis studies demonstrated that the Sp1-binding site that is located in the proximal promoter region plays an important role in transcriptional activity of the bovine Nanog promoter. The electrophoretic mobility shift assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. In addition, significant inhibition of Nanog promoter activity by the Sp1 mutant was observed in murine embryonic stem cells. Furthermore, chromatin-immunoprecipitation assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. These results suggest that Sp1 is an essential regulatory factor for bovine Nanog transcriptional activity.

• Journal of Microbiology and Biotechnology
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• v.18 no.2
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• pp.219-227
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• 2008

The free-living photoheterotrophic Gram-negative bacterium Rhodobacter sphaeroides possesses a quorum-sensing (QS) regulatory system mediated by CerR-CerI, a member of the LuxR-LuxI family. To identify the genes affected by the regulatory system, random lacZ fusions were generated in the genome of R. sphaeroides strain 2.4.1 using a promoter-trapping vector, pSG2. About 20,000 clones were screened and 23 showed a significantly different level of ${\beta}$-gal activities upon the addition of synthetic 7,8-cis-N-tetradecenoyl-homoserine lactone (RAI). Among these 23 clones, the clone showing the highest level of induction was selected for further study, where about a ten-fold increase of ${\beta}$-gal activity was exhibited in the presence of RAI and induction was shown to be required for cerR. In this clone, the lacZ reporter was inserted in a putative gene that exhibited a low homology with catD. A genetic analysis showed that the expression of the catD homolog was initiated from a promoter of another gene present upstream of the catD. This upstream gene showed a strong homology with luxR and hence was named qsrR (quorum-sensing regulation regulator). A comparison of the total protein expression profiles for the wild-type cells and qsrR-null mutant cells using two-dimensional gel electrophoresis and a MALDI-TOF analysis allowed the identification of sets of genes modulated by the luxR homolog.

• Microbiology and Biotechnology Letters
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• v.33 no.3
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• pp.189-193
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• 2005

IgE-dependent histamine-releasing factor(HRF) was initially described as a secretagogue for secretion of histamine from IgE+ basophils from a subset of allergic donors. Previously, we identified that S98 residue of HRF was phosphorylated using anti-HRFpS98 antibody which specifically recognizes the phosphorylated serine residue of HRF and HRFS98A mutant construct. In vitro kinase assay, only wild type HRF was phosphorylated by PKC, and S98A HRF was not affected by PKC. In this study, we attempted to characterize the phosphatase which specifically dephosphorylates HRF by immunoprecipitation and pull-down assay. In RBL-2H3 cells, HRF interacted only with calcineurin (also called as PP2B, calcium/calmodulin-dependent phosphatase) but not with PP1 or PP2A. The results suggest that HRF is most likely dephosphory-lated by calcineurin.

• Journal of Microbiology and Biotechnology
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• v.18 no.5
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• pp.828-836
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• 2008

The biocontrol rhizobacterium Pseudomonas sp. M18 can produce two kinds of antibiotics, namely pyoluteorin (Plt) and phenazine-1-carboxylic acid (PCA), and is antagonistic against a number of soilborne phytopathogens. In this study, a luxR-type quorum-sensing regulatory gene, vqsR, was identified and characterized immediately downstream of the Plt gene cluster in strain MI8. A vqsR-inactivated mutant led to a significant decrease in the production of Plt and its biosynthetic gene expression. However, this was restored when introducing the vqsR gene by cloning into the plasmid pME6032 in trans. The vqsR mutation did not exert any obvious influence on the production of PCA and its biosynthetic gene expression and the production of N-acylhomoserine lactones (C4 and C8-HSLs) and their biosynthetic gene rhlI expression. Accordingly, these results introduce VqsR as a regulator of Plt production in Pseudomonas spp., and suggest that the regulatory mechanism of vqsR in strain M18 is distinct from that in P. aeruginosa. In addition, it was demonstrated that vqsR mutation did not have any obvious impact on the expression of Plt-specific ABC transporters and other secondary metabolic global regulators, including GacA, RpoS, and RsmA.

• Korean Journal of Microbiology
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• v.53 no.3
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• pp.149-155
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• 2017

The nitrogen phosphotransferase (PTS) system is a regulatory cascade present in most Proteobacteria, where it controls different functions. The nitrogen PTS is usually composed of $EI^{Ntr}$ (encoded by the ptsP gene), NPr (encoded by the ptsO gene), and $EIIA^{Ntr}$ (encoded by the ptsN gene). While $EIIA^{Ntr}$ plays a role in a variety of cellular processes, such as potassium homeostasis, regulation of ppGpp accumulation, nitrogen and carbon metabolisms, and regulation of ABC transporters, little information is available for a physiological role of NPr. A recent study showed that dephosphorylated NPr affects adaptation to envelope stresses in Escherichia coli. In this study, we provide another phenotype related to NPr. The ptsP mutant showed a filamentation phenotype. The filamentation phenotype of the ptsP mutant was recovered by additional deletion of the ptsO gene, but not by additional deletion of the ptsN gene, suggesting that an increased level of dephosphorylated NPr in the ptsP mutant renders cells the filamentous growth. This idea was confirmed by the fact that cells with increased levels of dephosphorylated NPr shows the filamentation phenotype. Additionally, we showed that cell size of E. coli increases with incremental dephosphorylated NPr concentrations. These results suggested that dephosphorylated NPr induces morphological change of E. coli.

• Journal of Life Science
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• v.17 no.5 s.85
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• pp.723-727
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• 2007

Resistance to metronidazole in Helicobacter pylori results from inactivation of rdxA and frxA, the chromosomal genes for a nitroreductase that normally converts metronidazole from prodrug to bactericidal agent. Two types of metronidazole susceptible strains had been found distinguishable by their apparent levels of frxA expression. Most common in the populations we had studied were strains that required only rdxA inactivation to become resistant to moderate levels of metronidazole(type I strains). The second strain type required inactivation of both frxA and rdxA to become resistance to metronidazole(type II strains): this was linked to a relatively high level of frxA gene transcription in the type II strains. The fdxA gene regulated fdxA as well as rdxA gene. Thus, to study the function of fdxA as a regulatory gene we constructed a null mutant of fdxA in H. pylori genome and identified over-and under-expressed proteins by fdxA using two-dimensional(2-D) electrophoresis and MALDI-TOP-MS. There were four over-expressed proteins in fdxA mutant; nifU-like protein(HP0221), frxA(HP0642), nonheme ferritin(HP0653), and hypothetical protein(HP0902). Three under-expressed proteins were also identified in fdxA mutant, including 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (HP0089), (3R)-hydroxymyristoyl ACP dehydratase(HP1376), and thioredoxin(HP1458).

• Journal of Microbiology
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• v.41 no.3
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• pp.259-261
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• 2003

The nsdD gene has been predicted to encode a GATA type transcription factor with the type IVb zinc finger DNA binding domain functions in activating sexual development of A. nidulans. In several allelic mutants of nsdD producing truncated NsdD polypeptides lacking the C-terminal zinc finger, the transcription level of nsdD gene was greatly increased. Also in an over-expressed mutant, the transcription under its own promoter was reduced. These results suggest that the expression of nsdD is negatively autoregulated. When the nsdD gene was over-expressed, cleistothecia were formed in excess amounts even in the presence of 0.6 M KC1 that inhibited sexual development of the wild type. Northern blot analysis revealed that the expression of nsdD was repressed by 0.6 M KC1. These results strongly suggest that the inhibition of sexual development by salts was carried out via the nsdD involved regulatory network.

• Journal of Microbiology
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• v.35 no.4
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• pp.277-283
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• 1997

Promoters inducible by paraquat, a superocide-generating agent, were isolated from Escherichia coli using a promoter-probing plasmid pRS415 with promoterless lacA gene. Twenty one promoters induced by paraquat were selected and further characterized. From sequence analysis, thirteen of the promoters were mapped to their specific loci on the Escherichia coli chromosome. Several promoters were mapped to the upstream of known genes such as usgl, katG, and mglB, whose relationships with superoxide response have not been previously reported. Other promoters were mapped to the upstream region of unknown open reading frames. Downstream of HC 96 promoter are uncharacterized ORFs whose sequences are homologous to ABC-transporter subunits. Downstream of HC84 promoter is an ORF encoding a transcriptional regulator-like protein, which contains a LysR family-specific HTH (helix-turn-helix) DNA bindign motif. We investigated whether these promoters belong to the soxRS regulon. All promoters except HC96 were found to belong to the soxRS regulon. The HC96 promoter was significantly induced by paraquat in the soxRS deletion mutant strain. The basal transcription level of three promoters (HE43, HC71, HD94) significantly increased at the stationary phase, implying that they are regulated by RpoS. However, paraquat inducibility of all promoters disappeared in the stationary phase, suggesting that SoxRS regulatory system is active only in rapidly growing cells.

• Journal of Microbiology
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• v.45 no.6
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• pp.492-498
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• 2007

GacS and GacA proteins form a two component signal transduction system in bacteria. Here, Tn5 transposon gacS and gacA (Gac) mutants of Pseudomonas sp. KL28, an alkylphenol degrader, were isolated by selecting for smooth colonies of strain KL28. The mutants exhibited reduced ability to migrate on a solid surface. This surface motility does not require the action of flagella unlike the well-studied swarming motility of other Pseudomonas sp. The Gac mutants also showed reduced levels of biofilm and pellicle formation in liquid culture. In addition, compared to the wild type KL28 strain, these mutants were more resistant to high concentrations of m-cresol but were more sensitive to $H_2O_2$, which are characteristics that they share with an rpoS mutant. These results indicate that the Gac regulatory cascade in strain KL28 positively controls wrinkling morphology, biofilm formation, surface translocation and $H_2O_2$ resistance, which are important traits for its capacity to survive in particular niches.

• Proceedings of the Korean Society of Applied Pharmacology
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• 2005.04a
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• pp.5-14
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• 2005

Recently, data from several groups have raised the concept of 'checkpoint' in transcription. As capping of nascent RNA transcript is tightly coupled to RNA polymerase II transcription, we seek to obtain direct evidence that transcripiton checkpoint via capping enzyme functions in this early regulatory step. One of temperature sensitive (ts) alleles of ceg1, a guanylyltransferase subunit of the Saccharomyces cerevisiaecapping enzyme, showed 6-azauracil (6AU) sensitivity at the permissive growth temperature, which is a phenotype that is correlated with a transcription elongational defect. This ts allele, ceg1-63 also has an impaired ability to induce PUR5 in response to a 6AU treatment. However, this cellular and molecular defect is not due to the preferential degradation of the transcript attributed from a lack of guanylyltransferase activity. On the contrary, the data suggests that the guanylyltransferase subunit of the capping enzyme plays a role in transcription elongation. First, in addition to the 6AU sensitivity, ceg1-63is synthetically lethal with elongation defective mutations of the largest subunit of RNA polymerase II. Secondly, it exhibited a lower GAL1 mRNA turn-over after glucoseshut off. Third, it decreased the transcription read through a tandem array of promoter proximal pause sites in an orientation dependent manner. Interestingly, this mutant also showed lower pass through a pause site located further downstream of the promoter. Taken together, these results suggest that the capping enzyme plays the role of an early transcription checkpoint possibly in the step of the reversion of repression by stimulating polymerase to escape from the promoter proximal arrest once RNA becomes appropriately capped.