• The Korean Journal of Physiology and Pharmacology
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• 제3권2호
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• pp.157-164
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• 1999

To determine molecular mechanisms of Aquaporin-CD (AQP2) gene regulation, the promoter region of the AQP2 gene was examined by transiently transfecting a promoter-luciferase reporter fusion gene into mouse renal collecting duct cell lines such as mIMCD-3, mIMCD-K2, and M-1 cells, and NIH3T3 mouse embryo fibroblast cells. PCR-Southern analysis reveals that mIMCD-3 and mIMCD-K2 cells express AQP2, but M-1 and NIH3T3 cells do not, and that the treatment with cpt-cAMP $(400\;{\mu}M)$) or forskolin/isobutylmethylxanthine (IBMX) increased the AQP2 expression in IMCD cells. In both IMCD and NIH3T3 cells, the constructs containing the promoter of AQP2 gene showed promoter activities, indicating lack of tissue-specific element in the 1.4 kb 5'-flanking region of the mouse AQP2 gene. Luciferase activity in the IMCD cells transfected with the construct containing 5-flanking region showed responsiveness to cpt-cAMP, indicating that the 1.4 kb 5'-flanking region contains the element necessary for the regulatory mechanism by cAMP. The promoter-luciferase constructs which do not have a cAMP-responsible element (CRE) still showed the cAMP responsiveness in IMCD cells, but not in NIH3T3 cells. Increase in medium osmolarity did not affect AQP2 promoter activity in mIMCD-K2 cells. These results demonstrate that AQP2 gene transcription is increased with cAMP treatment through multiple motifs including CRE in the 5'-flanking region of the gene in vitro, and the regulatory mechanism may be important for in vivo regulation of AQP2 expression.

• BMB Reports
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• 제47권11호
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• pp.619-624
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• 2014

Antisense RNA is a type of noncoding RNA (ncRNA) that binds to complementary mRNA sequences and induces gene repression by inhibiting translation or degrading mRNA. Recently, several small ncRNAs (sRNAs) have been identified in Escherichia coli that act as antisense RNA mainly via base pairing with mRNA. The base pairing predominantly leads to gene repression, and in some cases, gene activation. In the current study, we examined how the location of target sites affects sRNA-mediated gene regulation. An efficient antisense RNA expression system was developed, and the effects of antisense RNAs on various target sites in a model mRNA were examined. The target sites of antisense RNAs suppressing gene expression were identified, not only in the translation initiation region (TIR) of mRNA, but also at the junction between the coding region and 3' untranslated region. Surprisingly, an antisense RNA recognizing the upstream region of TIR enhanced gene expression through increasing mRNA stability.

• 대한수의학회지
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• 제46권4호
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• pp.355-361
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• 2006

Porcine epidemic diarrhea virus (PEDV) strain Chinju99, which was previously isolated from piglets suffering from severe diarrhea was used to characterize the membrane (M) protein gene to establish the molecular information, and the results will be useful in elucidating concepts related to molecular pathogenesis and antigenic structures of PEDV isolates. The Chinju99 M gene generated by reverse transcription and polymerase chain reaction (RT-PCR) consisted of 681 bases containing 22.3% adenine, 22.3% cytosine, 23.1% guanine and 32.3% thymine nucleotides, and the GC content was 45.4%. It had some nucleotide mismatches from M gene of other PEDV strains, such as CV777, Br1/87, KPEDV-9, JMe2, JS2004-2 and LJB-03 with 97-99% nucleotide sequence homology to these strains. Also, it encoded a protein of 226 amino acids, which had some mismatches from those of CV777, Br1/87, KPEDV-9, JMe2, JS20004-2 and LJB-03, as the amino acid sequence homology showed a 97-98% to these strains. The Chinju99 had a very close relationship to the Japanese strain JMe2 for the nucleotide and amino acid sequences of the M gene. The amino acids predicted from Chinju99 M gene consisted of mostly hydrophobic residues and contained three potential sites for asparagine (N)-linked glycosylation, two serine (S)-linked phosphorylation sites by protein kinase C, and two S- or threonine (T)-linked phosphorylation sites by casein kinase II.

• 미생물학회지
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• 제29권5호
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• pp.284-289
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• 1991

the nucleotide sequence of the 3' terminal 568 bases of the 18S rRNA gene from Mucor racemosus was determined. The 3' end of the structural gene was identified by comparison with the published sequence for the Saccharomyces cerevisiae gene. The M. racemosus gene was found to share 83.8% homology with that of S. cerevisiae and 71-81% homology with those of human, mouse, maize, Xenopus laevis and Tetrahymena thermophila. The known methylation sites in X. laevis and human were also highly conserved in M. racemosus and located within most conserved regions of 18S RNA gene throughout evolution.

• Journal of Microbiology and Biotechnology
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• 제27권4호
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• pp.808-815
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• 2017

We demonstrated the quantitative detection of a toxin-producing Microcystis aeruginosa (M. aeruginosa) strain with the laboratory protocol of the NanoGene assay. The NanoGene assay was selected because its laboratory protocol is in the process of being transplanted into a portable system. The mcyD gene of M. aeruginosa was targeted and, as expected, its corresponding fluorescence signal was linearly proportional to the mcyD gene copy number. The sensitivity of the NanoGene assay for this purpose was validated using both dsDNA mcyD gene amplicons and genomic DNAs (gDNA). The limit of detection was determined to be 38 mcyD gene copies per reaction and 9 algal cells/ml water. The specificity of the assay was also demonstrated by the addition of gDNA extracted from environmental algae into the hybridization reaction. Detection of M. aeruginosa was performed in the environmental samples with environmentally relevant sensitivity (${\sim}10^5$ algal cells/ml) and specificity. As expected, M. aeruginosa were not detected in nonspecific environmental algal gDNA over the range of $2{\times}10^0$ to $2{\times}10^7$ algal cells/ml.

• Molecules and Cells
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• 제24권3호
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• pp.351-357
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• 2007

Regions (about 3.7-3.8 kb) of the mitochondrial genomes (rrnL-cox1) of two tardigrades, a heterotardigrade, Batillipes pennaki, and a eutardigrade, Pseudobiotus spinifer, were sequenced and characterized. The gene order in Batillipes was $\underline{rrnL}-\underline{V}-\underline{rrnS}-\underline{Q}-\underline{I}$-M-nad2-W-$\underline{C}-\underline{Y}$-cox1, and in Pseudobiotus it was $\underline{rrnL}-\underline{V}-\underline{rrnS}-\underline{Q}$-M-nad2-W-$\underline{C}-\underline{Y}$-cox1. With the exception of the trnI gene, the two tardigrade regions have the same gene content and order. Their gene orders are strikingly similar to that of the chelicerate Limulus polyphemus (rrnL-V-rrnS-CR-I-Q-M-nad2-W-C-Y-cox1), which is considered to be ancestral for arthropods. Although the tardigrades do not have a distinct control region (CR) within this segment, the trnI gene in Pseudobiotus is located between rrnL-trnL1 and trnL2-nad1, and the trnI gene in Batillipes is located between trnQ and trnM. In addition, the 106-bp region between trnQ and trnM in Batillipes not only contains two plausible trnI genes with opposite orientations, but also exhibits some CR-like characteristics. The mitochondrial gene arrangements of 183 other protostomes were compared. 60 (52.2%) of the 115 arthropods examined have the M-nad2-W-C-Y-cox1 arrangement, and 88 (76.5%) the M-nad2-W arrangement, as found in the tardigrades. In contrast, no such arrangement was seen in the 70 non-arthropod protostomes studied. These are the first non-sequence molecular data that support the close relationship of tardigrades and arthropods.

• Journal of Microbiology and Biotechnology
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• 제18권2호
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• pp.228-233
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• 2008

Effects of chaperones on mRNA stability and gene expression were studied in order to develop an efficient Escherichia coli expression system that can maximize gene expression. The stability of mRNA was modulated by introducing various secondary structures at the 5'-end of mRNA. Four vector systems providing different 5'-end structures were constructed, and genes encoding GFPuv and endoxylanase were cloned into the four vector systems. Primer extension assay revealed different mRNA half-lives depending on the 5'-end secondary structures of mRNA. In addition to the stem-loop structure at the 5'-end of mRNA, coexpression of dnaK-dnaJ-grpE or groEL-groES, representative heat-shock genes in E. coli, increased the mRNA stability and the level of gene expression further, even though the degree of stabilization was varied. Our work suggests that some of the heat-shock proteins can function as mRNA stabilizers as well s protein chaperones.

• Toxicological Research
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• 제16권2호
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• pp.95-100
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• 2000

In previous study, both constitutive expression and 3-methylcholanthrene (3MC)-mediated elevation of CYP1A2 mRNA were demonstrated in human hepatoma HepG2 cells by reverse transcription-polymerase chain reaction (RT-PCR), suggesting that HepG2 cells would be appropriate for the study of human CYP1A2 regulation(Chung and Bresnick, 1994). Further studies were conducted to determine the basis of this induction phenomenon that is observed in HepG2 cells. Since CYP1A1 gene, another polycyclic hydrocarbon(PH)-inducible gene, is regulated by PHs through their interactions via receptors with cis-elements, the 5'-flanking region of human CYP 1A2 gene was analyzed to search such responsive elements. The promoter activity of various lengths of CYP1A2 gene sequence (-3203/+58bp) was measured in transiently-transfected HepG2 cells by fusion constructs containing the CAT, hGH or luciferase genes as a reporter. This region of the CYP1A2 gene, although containing a XRE, was only weakly responsive (less than 2 fold induction) to 10 nM of TCDD or 1 $\mu$M 3 MC treatment. This small enhancement of promoter activity is inconsistent with the previous observation, i.e., 12 to 14 fold-enhanced CYP1A2 mRNA from 1 $\mu$M 3 MC treated HepG2 cells, suggesting that additional mechanisms would exist for PH-mediated induction of CYP1A2 in these cells.

• BMB Reports
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• 제29권1호
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• pp.88-91
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• 1996

The anaerobically expressed gene aeg-46.5, which had been identified by the operon fusion technique with a hybrid bacteriophage of ${\lambda}$ and Mu, ${\lambda}$placMu53, was studied for its expression pattern and growth. The expression of aeg-46.5 was studied in the wild-type cell and mutant cells that have mutation (s) in the control gene of anaerobic respiration (fnr) and nitrate response (narL and narP). The ${\beta}$-galactosidase reporter gene showed maximum expression in narL host after two hours of aerobic to anaerobic switch in M9-Glc-nitrate medium. Both 40 mM and 100 mM concentrations of nitrate ion in the medium had little effect on expression level. We propose that aeg-46.5 is subject to multiple regulations of anaerobic activation by Fnr, nitrate activation by NarP and repression mediated by NarL.

• Asian Pacific Journal of Cancer Prevention
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• 제13권4호
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• pp.1557-1561
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• 2012

Objective: To investigate the promoter methylation status of the E-cadherin gene in non-small cell lung cancer (NSCLC) and its association with clinical pathological parameters, and to explore the relationship between downregulation of E-cadherin gene expression and the methylation status of its promoter region. Methods: Nested methylation-specific PCR was performed to examine CpG methylation within the 5' CpG island of the E-cadherin gene in lung cancer and para-cancerous tissue from 37 patients with primary non-small cell lung cancer. Quantitative real-time PCR was performed to measure the level of E-cadherin mRNA. Results: Of thirty-seven cases, 12 (32.4%) samples showed aberrant CpG methylation in tumor tissues compared with the corresponding normal tissues. In addition, a reduction in E-cadherin mRNA levels was observed in 11 of the 12 (91.7%) tumor tissues carrying a methylated E-cadherin gene. However, only 10 (43.5%) cases displayed reduced mRNA levels in tumor tissues from the remaining 23 cases (excluding 2 samples from which mRNA was unavailable) without methylation events. Downregulation of E-cadherin gene expression significantly correlated with the promoter methylation status of this gene. Conclusion: These results provide strong evidence that the methylation status of E-cadherin gene contributes to a reduction in the expression of E-cadherin mRNA, and may play a role in the development and progression of NSCLC.