• Korean Journal of Poultry Science
• /
• v.46 no.1
• /
• pp.17-24
• /
• 2019

Chickens have been considered as well-defined animal bioreactor. The optimized ovalbumin promoter is essential for recombinant protein production in transgenic chicken. Here we try to compare the activity and identify the effect of estrogen on ovalbumin promoter according to each promoter length with estrogen response element (ERE) existence. We cloned two (2.8 and 5.5 kb) ovalbumin promoters that the 5.5 kb contained the ERE but the 2.8 kb did not, and these two promoters were cloned to pGL4.11 vector. Additionally, we constructed another pGL4.11 vector containing of the 4.4 kb (with ERE) ovalbumin promoter deleted with 1 kb between ERE region and the 2.8 kb promoter. For reporter assay, HeLa, MES-SA, LMH/2A, and cEF cells were transfected with all the pGL4.11 vectors. The comparative analysis showed that the mutated 4.4 kb promoter has more potent activity than the 2.8 and 5.5 kb promoters in HeLa, MES-SA, and LMH/2A cells. However, there is no significant difference in cEFs. Also, these cells transfected with the mutated 4.4 kb promoter were treated with the $17{\beta}$-estradiol (0~3,000 nM) and HeLa, MES-SA, and LMH/2A cells showed estrogen responsibilities, but cEFs did not. Besides, the mutated 4.4 kb promoter has still higher activity than the 2.8 and 5.5 kb promoter, and there is no transcriptional induction effect in 2.8 kb promoter at 500 nM estrogen that is blood concentration of laying hens. Hence our study strongly suggested that the mutated 4.4 kb promoter is considered as one of the most efficient length for generating transgenic chicken.

• Genomics & Informatics
• /
• v.8 no.4
• /
• pp.206-211
• /
• 2010

The Fas (TNF receptor superfamily, member 6) (FAS)/FAS ligand (FASLG) interaction plays a central role in the regulation of programmed cell death. FAS and FASLG polymorphisms in promoter regions affect transcriptional activities. To investigate whether FAS and FASLG polymorphisms are associated with the development and clinical phenotypes of stroke, 2 promoter single nucleotide polymorphisms (SNPs) in FAS (rs1800682, -670C/T) and FASLG (rs763110, -844C/T) were selected and genotyped by direct sequencing in 220 stroke patients [107 ischemic stroke (IS), 77 intracerebral hemorrhage (ICH), and 36 subarachnoid hemorrhage (SAH)] and 369 control subjects. For the analysis of clinical symptoms, all stroke patients were divided into 3 clinical phenotypes according to the respective results of the National Institutes of Health Stroke Survey (NIHSS) and the Modified Barthel Index (MBI) and the presence or absence of complex regional pain syndrome (CRPS). The SNPStats, SNPAnalyzer, and Helixtree programs were used to analyze the genetic data. Multiple logistic regression models (codominant, dominant, and recessive) were used to estimate odds ratios (ORs), 95% confidence intervals (CIs), and p-values. The promoter SNP rs1800682 was associated with stroke in the codominant (OR=0.48, 95% CI=0.25-0.94, p=0.04) and dominant models (OR=0.51, 95% CI=0.30-0.87, p=0.011). However, a FASLG SNP (rs763110) was not in Hardy-Weinberg equilibrium (p<0.05). In the analysis of stroke types, rs1800682 was associated with IS in the codominant (OR=0.30, 95% CI=0.12-0.74, p=0.025), dominant (OR=0.44, 95% CI=0.23-0.88, p=0.018), and recessive models (OR=0.45, 95% CI=0.21-0.99, p=0.042). The genotype frequencies of rs1800682 were different between ICH and controls in the dominant model (OR=0.49, 95% CI=0.26-0.94, p=0.031) but not between SAH and controls. In the analysis of clinical symptoms, however, rs1800682 was not related to the 3 clinical phenotypes (NIHSS, MBI, and CRPS). These results suggest that a promoter SNP (rs1800682, -670C/T) in FAS may be associated with the development of stroke in the Korean population.

• Journal of Microbiology and Biotechnology
• /
• v.10 no.6
• /
• pp.829-835
• /
• 2000

The ptsH and ptsI genes of Lactococus lactis subsp. lactis ATCC 7962 (L. lactis 7962), encoding the general proteins of phosphotransferase system (PTS) components, HPr and enzyme I, respectively, were cloned and characterized. A 1.3 kb PCR product was obtained using a primer set that was hybridized to the internal region of the L. lactis 7962 pts HI genes and then subcloned into a low-copy number vector, pACYC184. The 5' upstream and 3' downstream region from the 1.3 kb fragment were subsequently clone using the chromosome walking method. The complete ptsHI operon was constructed and the nucleotide sequences determined. Two ORFs corresponding to HPr (88 amino acids) and enzyme I (575 amino acids) were located. The ptsHI genes of L. lactis 7962 showed a very high homology (84-90%) with those genes from other Gram-positive bacteria. A primer extension analysis showed that the transcription started at either one of two adjacent bases upstream of the start codon. Using a Northern analysis, two transcripts were detected; the first, a 0.3 kb transcript corresponding to ptsH and the second, a 2 kb transcript corresponding to ptsH and ptsI. The transcription level of ptsH was higher than that of ptsI. The concentration of the ptsH transcript in cells grown on glucose was similar to that in cells grown on lactose, yet higher than that in cells grown on galactose. The ptsI transcript was scarcely detected in cell grown on lactose or galactose. The ptsI transcript was scarcely detected in cells grown on lactose or galactose. The results of a sequence analysis and Northern blot confirmed that the ptsH and ptsI genes of L. lactis 7962 were arranged in an operon like other known ptsHI genes and the expression of the ptsHI genes was regulated at the transcriptional level in response to the carbon source.

• The Korean Journal of Mycology
• /
• v.47 no.4
• /
• pp.417-425
• /
• 2019

Biocontrol agents (BCAs) are widely used to protect plants from diverse biotic and abiotic stresses in agricultural and ecological fields. Among the various microbes, many subspecies of the gram-positive genus, Bacillus, have been successfully industrialized as eco-friendly biological pesticides and fertilizers. In the current study, we demonstrated that Bacillus thuringiensis C25 exhibited antagonistic effects on the mycelial growth of Rosellinia necatrix, a fungal phytopathogen. Scanning electron microscopy analysis revealed that B. thuringiensis C25 degraded the cell wall structures of R. necatrix mycelia. In the functional genomic analysis of B. thuringiensis C25, we annotated 5,683 genes and selected the gene sets that potentially encoded fungal cell wall degrading enzymes (CWDEs). The growth inhibition effects on R. necatrix were highly correlated with the transcriptional activity of the mycelial cell wall degrading genes of B. thuringiensis C25. The transcript levels of CWDEs, including CshiA, B, and Glycos_transf_2 genes in B. thuringiensis C25, were enhanced following co-cultivation with R. necatrix. In conclusion, our study suggested that B. thuringiensis C25 could serve as a suitable candidate for controlling R. necatrix and could facilitate elucidating the mechanisms underlying the antifungal activities of BCAs against phytopathogens.

• Molecules and Cells
• /
• v.28 no.4
• /
• pp.383-388
• /
• 2009

The dehydration responsive element binding protein 2C (DREB2C) is a dehydration responsive element/C-repeat (DRE/CRT)-motif binding transcription factor that induced by mild heat stress. Previous experiments established that overexpression of DREB2C cDNA driven by the cauliflower mosaic virus 35S promoter (35S:DREB2C) resulted in increased heat tolerance in Arabidopsis. We first analyzed the proteomic profiles in wild-type and 35S:DREB2C plants at a normal temperature ($22^{\circ}C$), but could not detect any differences between the proteomes of wild-type and 35S: DREB2C plants. The transcript level of DREB2C in 35S: DREB2C plants after treatment with mild heat stress was increased more than two times compared with expression in 35S:DREB2C plants under unstressed condition. A proteomic approach was used to decipher the molecular mechanisms underlying thermotolerance in 35S:DREB2C Arabidopsis plants. Eleven protein spots were identified as being differentially regulated in 35S:DREB2C plants. Moreover, in silico motif analysis showed that peptidyl-prolyl isomerase ROC4, glutathione transferase 8, pyridoxal biosynthesis protein PDX1, and elongation factor Tu contained one or more DRE/CRT motifs. To our knowledge, this study is the first to identify possible targets of DREB2C transcription factors at the protein level. The proteomic results were in agreement with transcriptional data.

• Korean Journal of Environmental Biology
• /
• v.40 no.3
• /
• pp.255-266
• /
• 2022

In crustaceans, molting is regulated by interactions between ecdysteroid and juvenile hormone (JH) signaling pathway-related genes. Unlike the ecdysteroid signaling pathway, little information on the role of JH signaling pathway-related genes in molting is available in zooplanktonic crustaceans. In this study, three genes (juvenile hormone acid O-methyltransferase (JHAMT), methoprene-tolerant (Met), and juvenile hormone epoxide hydrolase (JHEH)) which are involved in the synthesis, receptor-binding, and degradation of JH were identified using sequence and phylogenetic analysis in the brackish water flea, Diaphanosoma celebensis. Transcriptional changes in these genes during the molting cycle in D. celebensis were analyzed. Sequence and phylogenetic analysis revealed that these putative proteins may be functionally conserved along with those of insects and other crustaceans. In addition, the expression of the three genes was correlated with the molting cycle of D. celebensis, indicating that these genes may be involved in the synthesis and degradation of JH, resulting in normal molting. This study will provide information for a better understanding of the role of JH signaling pathway-related genes during the molting process in Cladocera.

• Animal Bioscience
• /
• v.37 no.4
• /
• pp.709-717
• /
• 2024

Objective: Abnormally increased somatic cell counts (SCCs) in milk is usually a sign of bovine subclinical mastitis. Mutual interaction between the host and its associated microbiota plays an important role in developing such diseases. The main objective of this study was to explore the difference between cows with elevated SCCs and healthy cattle from the perspective of host-microbe interplay. Methods: A total of 31 milk samples and 23 bovine peripheral blood samples were collected from Holstein dairy cattle to conduct an integrated analysis of transcriptomic and metagenomics. Results: The results showed that Ralstonia and Sphingomonas were enriched in cows with subclinical mastitis. The relative abundance of the two bacteria was positively correlated with the expression level of bovine transcobalamin 1 and uridine phosphorylase 1 encoding gene. Moreover, functional analysis revealed a distinct alternation in some important microbial biological processes. Conclusion: These results reveal the relative abundance of Ralstonia and Sphingomonas other than common mastitis-causing pathogens varied from healthy cows to those with subclinical mastitis and might be associated with elevated SCCs. Potential association was observed between bovine milk microbiota composition and the transcriptional pattern of some genes, thus providing new insights to understand homeostasis of bovine udder.

• 한국균학회소식:학술대회논문집
• /
• 2014.05a
• /
• pp.27-28
• /
• 2014

Beauveria bassiana (Cordycipitaceae, Hypocreales, Ascomycota) is an anamorphic fungus having a potential to be used as a biological control agent because it parasitizes a wide range of arthropod hosts including termites, aphids, beetles and many other insects. A number of bioactive secondary metabolites (SMs) have been isolated from B. bassiana and functionally verified. Among them, beauvericin and bassianolide are cyclic depsipeptides with antibiotic and insecticidal effects belonging to the enniatin family. Non-ribosomal peptide synthetases (NRPSs) play a crucial role in the synthesis of these secondary metabolites. NRPSs are modularly organized multienzyme complexes in which each module is responsible for the elongation of proteinogenic and non-protein amino acids, as well as carboxyl and hydroxyacids. A minimum of three domains are necessary for one NRPS elongation module: an adenylation (A) domain for substrate recognition and activation; a tholation (T) domain that tethers the growing peptide chain and the incoming aminoacyl unit; and a condensation (C) domain to catalyze peptide bond formation. Some of the optional domains include epimerization (E), heterocyclization (Cy) and oxidation (Ox) domains, which may modify the enzyme-bound precursors or intermediates. In the present study, we analyzed genomes of B. bassiana and its allied species in Hypocreales to verify the distribution of NRPS-encoding genes involving biosynthesis of beauvericin and bassianolide, and to unveil the evolutionary processes of the gene clusters. Initially, we retrieved completely or partially assembled genomic sequences of fungal species belonging to Hypocreales from public databases. SM biosynthesizing genes were predicted from the selected genomes using antiSMASH program. Adenylation (A) domains were extracted from the predicted NRPS, NRPS-like and NRPS-PKS hybrid genes, and used them to construct a phylogenetic tree. Based on the preliminary results of SM biosynthetic gene prediction in B. bassiana, we analyzed the conserved gene orders of beauvericin and bassianolide biosynthetic gene clusters among the hypocrealean fungi. Reciprocal best blast hit (RBH) approach was performed to identify the regions orthologous to the biosynthetic gene cluster in the selected fungal genomes. A clear recombination pattern was recognized in the inferred A-domain tree in which A-domains in the 1st and 2nd modules of beauvericin and bassianolide synthetases were grouped in CYCLO and EAS clades, respectively, suggesting that two modules of each synthetase have evolved independently. In addition, inferred topologies were congruent with the species phylogeny of Cordycipitaceae, indicating that the gene fusion event have occurred before the species divergence. Beauvericin and bassianolide synthetases turned out to possess identical domain organization as C-A-T-C-A-NM-T-T-C. We also predicted precursors of beauvericin and bassianolide synthetases based on the extracted signature residues in A-domain core motifs. The result showed that the A-domains in the 1st module of both synthetases select D-2-hydroxyisovalerate (D-Hiv), while A-domains in the 2nd modules specifically activate L-phenylalanine (Phe) in beauvericin synthetase and leucine (Leu) in bassianolide synthetase. antiSMASH ver. 2.0 predicted 15 genes in the beauvericin biosynthetic gene cluster of the B. bassiana genome dispersed across a total length of approximately 50kb. The beauvericin biosynthetic gene cluster contains beauvericin synthetase as well as kivr gene encoding NADPH-dependent ketoisovalerate reductase which is necessary to convert 2-ketoisovalarate to D-Hiv and a gene encoding a putative Gal4-like transcriptional regulator. Our syntenic comparison showed that species in Cordycipitaceae have almost conserved beauvericin biosynthetic gene cluster although the gene order and direction were sometimes variable. It is intriguing that there is no region orthologous to beauvericin synthetase gene in Cordyceps militaris genome. It is likely that beauvericin synthetase was present in common ancestor of Cordycipitaceae but selective gene loss has occurred in several species including C. militaris. Putative bassianolide biosynthetic gene cluster consisted of 16 genes including bassianolide synthetase, cytochrome P450 monooxygenase, and putative Gal4-like transcriptional regulator genes. Our synteny analysis found that only B. bassiana possessed a bassianolide synthetase gene among the studied fungi. This result is consistent with the groupings in A-domain tree in which bassianolide synthetase gene found in B. bassiana was not grouped with NRPS genes predicted in other species. We hypothesized that bassianolide biosynthesizing cluster genes in B. bassiana are possibly acquired by horizontal gene transfer (HGT) from distantly related fungi. The present study showed that B. bassiana is the only species capable of producing both beauvericin and bassianolide. This property led to B. bassiana infect multiple hosts and to be a potential biological control agent against agricultural pests.

• Tuberculosis and Respiratory Diseases
• /
• v.51 no.4
• /
• pp.315-324
• /
• 2001

Background : IL-8 is a potent chemotactic cytokine that plays an important role in the host defense mechanism against M. tuberculosis by recruiting inflammatory cells to the site of the infection. Lung epithelial cells, as well as alveolar macrophages are known to produce IL-8 in response to M. tuberculosis. IL-8 gene expression is mainly regulated on the level of transcription by NF-${\kappa}B$. This study investigated whether or not A549 cells produce IL-8 in NF-${\kappa}B$ dependent mechanism in response to macrophages phagocytosing M. tuberculosis. Methods : Peripheral blood monocytes that were obtained from healthy donors were cultured for 24 h with M. tuberculosis and a conditioned medium(CoMTB) was obtained. As a negative control, the conditioned medium without M. tuberculosis (CoMCont) was used. A549 cells were stimulated with M. tuberculosis, CoMCont and CoMTB and the IL-8 concentration in the culture media was measured by ELISA. The CoMTB induced IL-8 mRNA expression in the A549 cells was evaluated using RT-PCR, and CoMTB induced $I{\kappa}B{\alpha}$ degradation was measured using western blot analysis. CoMTB induced nuclear translocation and DNA binding of NF-${\kappa}B$ was also examined using an electrophoretic mobility shift assay(EMSA), and the CoMTB induced NF-${\kappa}B$ dependent IL-8 transcriptional activity was measured using a luciferase reporter gene assay. Results : CoMTB induced IL-8 production by A549 cells($46.8{\pm}4.8\;ng/ml$) was higher than with direct stimulation with M. tuberculosis ($6.8{\pm}2.9\;ng/ml$). CoMTB induced IL-8 mRNA expression increased after 2 h of stimulation and was sustained for 24 h. $I{\kappa}B{\alpha}$ was degraded after 10 min of CoMTB stimulation and reappeared by 60 min. CoMTB stimulated the nuclear translocation and DNA binding of NF-${\kappa}B$. The CoMTB induced NF-${\kappa}B$ dependent IL-8 transcriptional activity($13.6{\pm}4.3$ times control) was higher than either CoMCont($2.0{\pm}0.6$ times control) or M. tuberculosis ($1.4{\pm}0.6$ times control). Conclusion : A conditioned medium of peripheral blood monocytes phagocytosing M. tuberculosis stimulates NF-${\kappa}B$ dependent IL-8 production by the lung epithelial cells.

• Molecules and Cells
• /
• v.37 no.2
• /
• pp.178-186
• /
• 2014

Differential transcription of the clusterin (CLU) gene yields two CLU isoforms, a nuclear form (nCLU) and a secretory form (sCLU), which play crucial roles in prostate tumorigenesis. Pro-apoptotic nCLU and anti-apoptotic sCLU have opposite effects and are differentially expressed in normal and cancer cells; however, their regulatory mechanisms at the transcriptional level are not yet known. Here, we examined the transcriptional regulation of nCLU in response to hypoxia. We identified three putative hypoxia response elements (HREs) in the human CLU promoter between positions -806 and +51 bp. Using a luciferase reporter, electrophoretic gel mobility shift, and chromatin immunoprecipitation assays, we further showed that hypoxia-inducible factor-$1{\alpha}$ (HIF-$1{\alpha}$) bound directly to these sites and activated transcription. Exposure to the hypoxia-mimetic compound $CoCl_2$, incubation under 1% $O_2$ conditions, or overexpression of HIF-$1{\alpha}$ enhanced nCLU expression and induced apoptosis in human prostate cancer PC3M cells. However, LNCaP prostate cancer cells were resistant to hypoxia-induced cell death. Methylation-specific PCR analysis revealed that the CLU promoter in PC3M cells was not methylated; in contrast, the CLU promoter in LNCap cells was methylated. Co-treatment of LNCaP cells with $CoCl_2$ and a demethylating agent promoted apoptotic cell death through the induction of nCLU. We conclude that nCLU expression is regulated by direct binding of HIF-$1{\alpha}$ to HRE sites and is epigenetically controlled by methylation of its promoter region.