• Microbiology and Biotechnology Letters
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• v.30 no.1
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• pp.15-20
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• 2002

Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol (DAG) to phosphatidic acid and it may play a role in signal transduction in Escherichia coli as well as in eukaryotic cells. In addition, DGK is important for microorganisms to adapt to several physiological stimuli. In Bacillus subtilis, the effect of stress on dgk transcription was examined by northern hybridization. The high level of dgk transcription was induced against high osmolarity, low pH value and low temperature. Transcriptional analysis revealed that the dgk gene and dgk upstream locus (ORF2, ORF3 and ORF4) were transcribed as a polycistronic mRNA to form an approximately 2.5 kb transcript.

• Korean Journal of Microbiology
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• v.41 no.4
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• pp.300-305
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• 2005

A transcriptionally active fragment $(P_{19})$ isolated by utilizing the promoter-probe shuttle vector pSK1Cat was analyzed. By subcloning analysis, the 180 bp region $(P_{180})$ responsible for the activity was determined. Transcriptional fusion of the C. glutamicum metX gene to $P_{180}\;(P_{180}-metX)$ resulted in a 24-fold increase in MetX activity in a complex medium, while a 13-fold increase was observed with the $P_{tac}$ promoter. Additionally, the expression conferred by $P_{180}$ was not affected by methionine added to the growth medium, suggesting that the $P_{180}$ clone is useful for the deregulated expression of biosynthetic genes in C. glutamicum during amino acid fermentation. Introduction of $P_{180}-metX$ into a lysine-producing C. glutamicum resulted in the production of methionine to 0.8 g/l.

• Journal of Life Science
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• v.22 no.3
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• pp.417-427
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• 2012

We compared the transcriptome in response to propanol stress in wild-type and propanol-resistant mutant Escherichia coli using the DNA microarray technique. The correlation value of RNA expression between the propanol-treated wild type and the untreated-one was about 0.949, and 50 genes were differentially expressed by more than twofold in both samples. The correlation value of RNA expression between the propanol-treated mutant and the untreated one was about 0.951, and 71 genes in two samples showed differential expression patterns. However, the values between the wild type and mutant, regardless of propanol addition, were 0.974-0.992 and only 1-2 genes were differentially expressed in the two strains. The representative characteristics among differentially expressed genes in W3110 or P19 treated with propanol compared to untreated samples were up-regulation of hest shock response genes and down-regulation of genes relating to ribosome biosynthesis. In addition, many genes were regulated by transcription regulation factors such as ArcA, CRP, FNR, H-NS, GatR, or PurR and overexpressed by sigma factor RpoH. We confirmed that RpoH mediated an important host defense function in propanol stress in E. coli W3110 and P19 by comparison of cell growth rate among the wild type, rpoH disruptant mutant, and rpoH-complemented strain.

• Journal of Life Science
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• v.15 no.6 s.73
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• pp.895-903
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• 2005

Thrombospondin-1 (TSP-1), a negative regulator in tumor growth and angiogenesis, is cell-type specifically regulated under pathological conditions or by extracellular stimuli, and the regulation of TSP-1 gene expression is important for developing new approaches in tumor therapy. Mistletoe is a parasitir plant that have been used for immunomodulation and antitumor therapy. Helixor A is an aqueous part of mistletoes extract. Here we showed that TSP-1 expression was significantly induced at both mRNA and protein levels in the Hepatocarcinorna cell line (Hep3B) and primary bovine endothelial cell line (BAE) exposed to Helixor A. Our promoter analysis confirmed that the expression of TSP-1 gene was regulated by Helixor A at the transcriptional level. In cell invasion assay, the conditioned media obtained from treatment of these cells significantly reduced the number of invasive cells and also inhibited capillary-like tube formation of BAE cells on Matrigel. Moreover, the inhibitory efforts of the conditioned media on cell invasion and tube formation were reversed by blocking with anti-TSP-1 neutralizing antibodies, suggesting that TSP-1 is involved in Helixor A-indured antiangiogenic effect. Taken together, our results suggest that Helixor A have an antiangiogenic effects through upregulation of TSP-1.

• Korean Journal of Microbiology
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• v.53 no.4
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• pp.292-296
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• 2017

In eukaryote, THO/TREX complex plays a critical role in transcriptional elongation, pre-mRNA processing, and nuclear mRNA export. This complex is evolutionally well- conserved, but there are some differences in composition and function according to organisms. Here we showed that spTex1, a component of THO/TREX complex, is not essential for growth and mRNA export in a fission yeast, Schizosaccharomyces pombe, which is more similar to higher eukaryote than budding yeast. Deletion and overexpression of the spTex1 gene do not lead to any detectable growth phenotype and accumulation of poly(A)+ RNA in the nucleus. And the spTex1-GFP protein is localized mainly in the nucleus. Yeast two-hybrid and Co-immunoprecipitation analysis showed that the spTex1 protein interacted with spHpr1 (THOC1) and spTho2 (THOC2), main subunits of THO complex. We conclude that the S. pombe Tex1 is a component of THO/TREX complex, but does not plays important roles in growth and bulk mRNA export from the nucleus.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.195-203
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• 2018

Interferon-tau (IFNT) is known as a major conceptus protein that signals the process of maternal recognition of pregnancy in ruminants. Also, multiple interferon genes exist in cattle, However, molecular mechanisms of these bovine IFNT (bIFNT) genes whose expressions are limited have not been characterized. We and others have observed that expression levels of bovine subtype IFNT genes in the tissues of ruminants; thus, bIFNT1 and other new type I (bIFNTc1/c2/c3) gene co-exist during the early stages of conceptus development and non-trophoblast cells. Its genes transcription could be regulated through CDX2 and ETS2 and JUN and/or cAMP-response element binding protein (CREB)-binding protein (CREBBP) expression, a transcription factor implicated in the control of cell differentiation in the trophectoderm. Bovine ear-derived fibroblast cells, were co-transfected with luciferase reporter constructs carrying upstream (positions -1000 to +51) regions of bIFNT1 and other new type I gene and various transcription factor expression plasmids. Compared to each - 1kb-bIFNT1/c1/c2/c3-Luc increased when this constructs were co-transfected with CDX2, ETS2, JUN and/or CREBBP. Also, Its genes was had very effect on activity by CDX2, either alone or with the other transcription factors, markedly increased luciferase activity. However, the degree of transcriptional activation of the bIFNTc1 gene was not similar to that bIFNT1/c2/c3 gene by expression plasmid. Furthermore, Sequence analyses also revealed that the expression levels of bIFNT1/c2/c3 gene mRNAs expression were highest on day 17, 20 and 22 trophoblast and, Madin-Darby bovine kidney (MDBK), Bovine ear-derived fibroblast (EF), and endometrium (Endo) non-trophoblast cells. But, bIFNTc1 mRNA had not same expression level, bIFNTc1 lowest levels than those of IFNT1/c2/c3 gene in both trophoblast and non-trophoblast cells. These results demonstrate that bovine subtype bIFNT genes display differential, in the trophoblast and non-trophoblast cells.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.12
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• pp.1942-1949
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• 2019

Objective: Leukocyte cell-derived chemotaxin 2 (LECT2) is associated with several physiological processes including inflammation, tumorigenesis, and natural killer T cell generation. Chicken LECT2 (chLECT2) gene was originally identified as one of the differentially expressed genes in chicken kidney tissue, where the chickens were fed with different calcium doses. In this study, the molecular characteristics and gene expression of chLECT2 were analyzed under the stimulation of toll-like receptor 3 (TLR3) ligand to understand the involvement of chLECT2 expression in chicken metabolic disorders. Methods: Amino acid sequence of LECT2 proteins from various species including fowl, fish, and mammal were retrieved from the Ensembl database and subjected to Insilco analyses. In addition, the time- and dose-dependent expression of chLECT2 was examined in DF-1 cells which were stimulated with polyinosinic:polycytidylic acid (poly [I:C]), a TLR3 ligand. Further, to explore the transcription factors required for the transcription of chLECT2, DF-1 cells were treated with poly (I:C) in the presence or absence of the nuclear factor ${\kappa}B$ ($NF{\kappa}B$) and activated protein 1 (AP-1) inhibitors. Results: The amino acid sequence prediction of chLECT2 protein revealed that along with duck LECT2 (duLECT2), it has unique signal peptide different from other vertebrate orthologs, and only chLECT2 and duLECT2 have an additional 157 and 161 amino acids on their carboxyl terminus, respectively. Phylogenetic analysis suggested that chLECT2 is evolved from a common ancestor along with the actinopterygii hence, more closely related than to the mammals. Our quantitative polymerase chain reaction results showed that, the expression of chLECT2 was up-regulated significantly in DF-1 cells under the stimulation of poly (I:C) (p<0.05). However, in the presence of $NF{\kappa}B$ or AP-1 inhibitors, the expression of chLECT2 is suppressed suggesting that both $NF{\kappa}B$ and AP-1 transcription factors are required for the induction of chLECT2 expression. Conclusion: The present results suggest that chLECT2 gene might be a target gene of TLR3 signaling. For the future, the expression pattern or molecular mechanism of chLECT2 under stimulation of other innate immune receptors shall be studied. The protein function of chLECT2 will be more clearly understood if further investigation about the mechanism of LECT2 in TLR pathways is conducted.

• Animal Bioscience
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• v.34 no.2
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• pp.172-184
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• 2021

Objective: Vanin1 (VNN1) is a pantetheinase that can catalyze the hydrolysis of pantetheine to produce pantothenic acid and cysteamine. Our previous studies showed that VNN1 is specifically expressed in chicken liver. In this study, we aimed to investigate the roles of peroxisome proliferators activated receptor α (PPARα) and miRNA-181a-5p in regulating VNN1 gene expression in chicken liver. Methods: 5'-RACE was performed to identify the transcription start site of chicken VNN1. JASPAR and TFSEARCH were used to analyze the potential transcription factor binding sites in the promoter region of chicken VNN1 and miRanda was used to search miRNA binding sites in 3' untranslated region (3'UTR) of chicken VNN1. We used a knock-down strategy to manipulate PPARα (or miRNA-181a-5p) expression levels in vitro to further investigate its effect on VNN1 gene transcription. Luciferase reporter assays were used to explore the specific regions of VNN1 targeted by PPARα and miRNA-181a-5p. Results: Sequence analysis of the VNN1 promoter region revealed several transcription factor-binding sites, including hepatocyte nuclear factor 1α (HNF1α), PPARα, and CCAAT/enhancer binding protein α. GW7647 (a specific agonist of PPARα) increased the expression level of VNN1 mRNA in chicken primary hepatocytes, whereas knockdown of PPARα with siRNA increased VNN1 mRNA expression. Moreover, the predicted PPARα-binding site was confirmed to be necessary for PPARα regulation of VNN1 gene expression. In addition, the VNN1 3'UTR contains a sequence that is completely complementary to nucleotides 1 to 7 of miRNA-181a-5p. Overexpression of miR-181a-5p significantly decreased the expression level of VNN1 mRNA. Conclusion: This study demonstrates that PPARα is an important transcriptional activator of VNN1 gene expression and that miRNA-181a-5p acts as a negative regulator of VNN1 expression in chicken hepatocytes.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.66 no.4
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• pp.365-374
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• 2021

Soybean isoflavones are essential secondary metabolites synthesized through the phenylpropanoid pathway, and they play vital roles in human health. Isoflavone content is a complex quantitative trait controlled by multiple genes, and the genetic mechanisms underlying isoflavone biosynthesis remain largely unknown. Therefore, the present study analyzed the content of isoflavone and expression of six key genes involved in its biosynthesis (i.e., CHS6, HID, IF7GT, IF7MaT, GmIMaT1, and GmIMaT3) during soybean seed germination. Isoflavone content was quantified using high-performance liquid chromatography, and isoflavone biosynthetic gene expression was analyzed using quantitative real-time PCR. Two cultivars, namely 'Daepung2ho' and 'Pungsannamulkong', which are high- and low-isoflavone cultivars, respectively, were used. Isoflavone accumulation gradually increased with the progression of the germination period. As such, malonyl glucosides accounted for over 80% of the total content, whereas acetyl glucosides were present at trace amounts. Transcriptional analysis of isoflavone biosynthetic genes demonstrated expression patterns parallel to isoflavone content; however, there was no clear correlation between isoflavone content and gene expression. Moreover, most isoflavone biosynthetic genes showed different expression patterns depending on the individual gene or genotypes. Among the tested genes, HID showed consistently higher expression, except at 3 days after germination, and its expression was upregulated in 'Daepung2ho' but downregulated in 'Pungsannamulkong'. In addition, all tested genes exhibited different expression patterns between cotyledons and hypocotyls and responded differently to the germination period. These findings suggest that the expression levels of isoflavone biosynthetic genes are not consistent with the germination period and appear to be genotype-dependent.

• Animal Bioscience
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• v.35 no.11
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• pp.1787-1799
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• 2022

Objective: Choline deficiency, one main trigger for nonalcoholic fatty liver disease (NAFLD), is closely related to lipid metabolism disorder. Previous study in a choline-deficient model has largely focused on gene expression rather than gene structure, especially sparse are studies regarding to alternative splicing (AS). In modern life science research, primary hepatocytes culture technology facilitates such studies, which can accurately imitate liver activity in vitro and show unique superiority. Whereas limitations to traditional hepatocytes culture technology exist in terms of efficiency and operability. This study pursued an optimization culture method for duck primary hepatocytes to explore AS in choline-deficient model. Methods: We performed an optimization culture method for duck primary hepatocytes with multi-step digestion procedure from Pekin duck embryos. Subsequently a NAFLD model was constructed with choline-free medium. RNA-seq and further analysis by rMATS were performed to identify AS events alterations in choline-deficency duck primary hepatocytes. Results: The results showed E13 (embryonic day 13) to E15 is suitable to obtain hepatocytes, and the viability reached over 95% by trypan blue exclusion assay. Primary hepatocyte retained their biological function as well identified by Periodic Acid-Schiff staining method and Glucose-6-phosphate dehydrogenase activity assay, respectively. Meanwhile, genes of alb and afp and specific protein of albumin were detected to verify cultured hepatocytes. Immunofluorescence was used to evaluate purity of hepatocytes, presenting up to 90%. On this base, choline-deficient model was constructed and displayed significantly increase of intracellular triglyceride and cholesterol as reported previously. Intriguingly, our data suggested that AS events in choline-deficient model were implicated in pivotal biological processes as an aberrant transcriptional regulator, of which 16 genes were involved in lipid metabolism and highly enriched in glycerophospholipid metabolism. Conclusion: An effective and rapid protocol for obtaining duck primary hepatocytes was established, by which our findings manifested choline deficiency could induce the accumulation of lipid and result in aberrant AS events in hepatocytes, providing a novel insight into various AS in the metabolism role of choline.