• Korean Journal of Microbiology
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• v.52 no.3
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• pp.249-253
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• 2016

The inky cap, Coprinellus congregatus, produces several laccase isozymes during its life cycle: both hyphal tip laccase and sclerotial laccase are involved in the fungal development. When this fungus was transferred to an acid liquid medium (pH 4.0-4.5), a new laccase was synthesized and secreted into the culture supernatant. In order to examine its regulation by external pH, green fluorescent protein gene was ligated at the downstream of the promoters having different lengths. These expression vectors having different promoter lengths were inserted into the fungal transformation vector, pBARGEM7-1. These expression vectors were introduced to the mating type a1 and a2 monokaryons, and the transformants were selected by the phosphinothricin resistance. Transformant a1 (a1TF) and transformant a2 (a2TF) were mated with each other to generate homozygotic dikaryon transformants. All these transformants were grown in neutral liquid medium for 5 days, and then the whole cell homogenates were transferred to the acidic liquid medium (pH 4.1). After 36 h incubation at $25^{\circ}C$, cells were harvested for the analysis of GFP expression. GFP expression was detected in the transformant having full-length promoter (2.0 kb), but other transformants having shorter length promoter (shorter than 1.29 kb) failed to show the fluorescence. Therefore, the acid-responsive element in the laccase promoter should be localized between -2.0 kb ~ -1.29 kb region.

• Asian-Australasian Journal of Animal Sciences
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• v.28 no.12
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• pp.1721-1728
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• 2015

Embryonic stem cells (ESCs) have been used as a powerful tool for research including gene manipulated animal models and the study of developmental gene regulation. Among the critical regulatory factors that maintain the pluripotency and self-renewal of undifferentiated ESCs, NANOG plays a very important role. Nevertheless, because pluripotency maintaining factors and specific markers for livestock ESCs have not yet been probed, few studies of the NANOG gene from domestic animals including bovine have been reported. Therefore, we chose mouse ESCs in order to understand and compare NANOG expression between bovine, human, and mouse during ESCs differentiation. We cloned a 600 bp (-420/+181) bovine NANOG 5'-flanking region, and tagged it with humanized recombinant green fluorescent protein (hrGFP) as a tracing reporter. Very high GFP expression for bovine NANOG promoter was observed in the mouse ESC line. GFP expression was monitored upon ESC differentiation and was gradually reduced along with differentiation toward neurons and adipocyte cells. Activity of bovine NANOG (-420/+181) promoter was compared with already known mouse and human NANOG promoters in mouse ESC and they were likely to show a similar pattern of regulation. In conclusion, bovine NANOG 5-flanking region functions in mouse ES cells and has characteristics similar to those of mouse and human. These results suggest that bovine gene function studied in mouse ES cells should be evaluated and extrapolated for application to characterization of bovine ES cells.

• Biomedical Science Letters
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• v.23 no.3
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• pp.272-276
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• 2017

HOX genes are transcription factors that play important roles in body patterning and cell fate specification during normal development. In previous study, we found aberrant overexpression of HOXB5 in breast cancer tissues and cell lines, and demonstrated that HOXB5 is important in regulation of cell proliferation, tamoxifen resistance, and invasiveness through the epithelial-mesenchymal transition (EMT). Although the relationship between HOXB5 and phenotypic changes in MCF7 breast cancer cells has been studied, the molecular function of HOXB5 as a transcription factor remains unclear. IL-6 has been reported to be involved in not only inflammation but also cancer progression, which is characterized by the increase of growth speed and invasiveness of tumor cells. In this study, we selected Interleukin-6 (IL-6) as HOXB5 putative downstream target gene and discovered that HOXB5 transcriptionally up-regulated the expression of IL-6 in HOXB5 overexpressing MCF7 cells. The upstream region (~1.2 kb) of IL-6 promoter turned out to contain several putative HOX consensus binding sites. Chromatin immunoprecipitation assay confirmed that HOXB5 directly binds to the promoter region of IL-6 and positively regulated the expression of IL-6. These data all together, indicate that HOXB5 promotes IL-6 transcription by actively binding to the putative binding sites located in the upstream region of IL-6, which enable to increase its promoter activity in MCF7 breast cancer cells.

• Parasites, Hosts and Diseases
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• v.48 no.4
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• pp.291-295
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• 2010

The onset, severity, and ultimate outcome of malaria infection are influenced by parasite-expressed virulence factors and individual host responses to these determinants, In both humans and mice, liver injury is involved after parasite entry, which persists until the erythrocyte stage after infection with the fatal strain Plasmodium falciparum (Pf), Hepatocyte growth factor (HGF) has strong anti-apoptotic effects in various kinds of cells, and also has diverse metabolic functions. In this work, Pf-subtilisin-like protease 2 (Pf-Sub2) 5' untranslated region (UTR) was analyzed and its transcriptional activity was estimated by luciferase expression. Fourteen TATA boxes were observed but only one Oct-1 and c-Myb were done. In addition, host HGF interaction with Pf-Sub2 was evaluated by co-transfection of HGF- and Pf-Sub2-cloned vector. Interestingly, -1,422/+12 UTR exhibited the strongest luciferase activity but -329 to + 12 UTR did not exhibit luciferase activity. Moreover, as compared with the control of unexpressed HGF, the HGF protein suppressed luciferase expression driven by the 5' untranslated region of the Pf-Sub2 promoter. Taken together, it is suggested that HGF controls and interacts with the promoter region of the Pf-Sub2 gene.

• The Journal of the Korean Society for Microbiology
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• v.35 no.3
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• pp.215-224
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• 2000

In this study, the distribution of the mec regulator genes and the presence of the mutation in mecI gene and mec promoter region among 50 MRSA clinical isolates derived from a single university hospital in Korea were analyzed. Among 50 MRSA strains, 13 strains had a deletion of mecI gene, and 37 strains were found to have mutations in mecI gene or mecA promoter region corresponding to a presumptive operator of mecA, i.e., the binding site of the repressor protein. Furthermore, in order to track the evolution of methicillin-resistant Staphylococcus aureus (MRSA) distributed in Korea, we determined the MRSA clonotype by combined use of genetic organization patterns of mec regulator genes, ribotype, and coagulase type. As the result, 48 of 50 MRSA strains could be classified into four distinct clones. Clonotype I is characterized by the coagulase type 3, deletion of mecI gene, and ribotype 1 shared by NCTC10442, the first reported MRSA isolate in England (9 strains). Clonotype II is characterized by the coagulase type 4, C to T substitution at position 202 of mecI gene, and ribotypes 2, 3 and 4 shared by 85/3619 strain isolated in Austria (10 strains). Clonotype III is characterized by the coagulase type 2, mutations of mecA promoter region and/or mecI, and ribotypes 4, 5, and 6 shared by N315 strain isolated in Japan (25 strains). Clonotype IV is characterized by the coagulase type 4, deletion of mecI gene, and ribotype 7 (4 strains). The clonality of two strains could not be determined due to their undefined ribotype.

• Journal of Microbiology and Biotechnology
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• v.20 no.2
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• pp.375-382
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• 2010

The available techniques for heterologous protein secretion in Lactobacillus strains are limited. The aim of the present study was to develop an efficient protein-secretion system using recombinant lactobacilli for various applications such as live delivery of biotherapeutics. For the construction of expression vectors, the Lactobacillus brevis slpA promoter, Lactobacillus casei prtP signal sequence, and mouse IL-10 sequences were used as a model system. Interestingly, the slpA promoter exhibited strong activity in L. casei, contrary to previous observations. In order to stabilize replication of the plasmid in E. coli, a removable terminator sequence was built into the promoter region. For the improvement of secretion efficiency, a DTNSD oligopeptide was added to the cleavage site of signal peptidase. The resulting plasmids provided remarkably efficient IL-10 secretion. Accumulation of the protein in the culture supernatant varied widely according to the pH conditions. By analysis of the secreted protein, formation of homodimers, and biological activity, IL-10 was confirmed to be functional. The presently constructed plasmids could be useful tools for heterologous protein secretion in L. casei.

• BMB Reports
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• v.53 no.10
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• pp.521-526
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• 2020

Endogenous retroviruses (ERVs) are retrotransposons present in various metazoan genomes and have been implicated in metazoan evolution as well as in nematodes and humans. The long terminal repeat (LTR) retrotransposons contain several regulatory sequences including promoters and enhancers that regulate endogenous gene expression and thereby control organismal development and response to environmental change. ERVs including the LTR retrotransposons constitute 8% of the human genome and less than 0.6% of the Caenorhabditis elegans (C. elegans) genome, a nematode genetic model system. To investigate the evolutionarily conserved mechanism behind the transcriptional activity of retrotransposons, we generated a transgenic worm model driving green fluorescent protein (GFP) expression using Human endogenous retroviruses (HERV)-K LTR as a promoter. The promoter activity of HERV-K LTR was robust and fluorescence was observed in various tissues throughout the developmental process. Interestingly, persistent GFP expression was specifically detected in the adult vulva muscle. Using deletion constructs, we found that the region from positions 675 to 868 containing the TATA box was necessary for promoter activity driving gene expression in the vulva. Interestingly, we found that the promoter activity of the LTR was dependent on che-1 transcription factor, a sensory neuron driver, and lin-15b, a negative regulator of RNAi and germline gene expression. These results suggest evolutionary conservation of the LTR retrotransposon activity in transcriptional regulation as well as the possibility of che-1 function in non-neuronal tissues.

• Journal of Crop Science and Biotechnology
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• v.10 no.1
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• pp.50-56
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• 2007

Barley S-adenosylmethionine synthetase1 gene, which was differentially expressed in seed development of extra early barley, was regulated by the phytohormones and abiotic stresses. In order to identify the regulation regions which were involved in transcriptional control of the phytohormones and abiotic stresses, we isolated 1459 bp fragment of HvSAMS1 gene promoter using genome walking strategy and deletion series were constructed. Deleted upstream fragments(-1459, -1223, -999, -766, -545, -301 bp) were fused to the GUS reporter gene and evaluated via Agrobacterium-mediated transient expression assay. Increased GUS activity of HvSMAS1 promoter -301/GUS construct under each of NaCl, $GA_3$, ABA and ethylene application was found. However, GUS activity was negligible in the leaves transformed with the HvSMAS1 promoter(-1459, -1223, -999, -766 and -545)/GUS constructs. No significant induction of GUS activity was observed for the ethionine and spermidine treatments. In order to locate promoter sequence of the HvSAMS1 gene that was critical for the activation of gene expression, deletion and addition promoter derivatives(+, includes 43 bp of 5' ORF) of the HvSAMS1 gene fused to the GUS reporter gene were applied. The tobacco leaves which harbored the additional HvSAMS1 promoter(-1459+, -1459 to -546, -545+ and -301+)/GUS construct did not significantly induce GUS activity as compared to the HvSAMS1 promoter(-1459, -545 and -301)/GUS constructs under each of NaCl, ABA and $GA_3$ treatment. However, the GUS activity was high in the tobacco leaves which harboring the -211 to -141 regions of the HvSAMS1 promoter. This result suggested that HvSAMS1 gene expression might be regulated by this region(from -211 to -141).

• KSBB Journal
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• v.10 no.1
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• pp.89-96
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• 1995

An Avabidofsis thaliana gene encoding phosphoribosyl anthranilate transferase is shown to be the gene that is defective in blue fluorescent trp 1 mutant plants. This gene, named PAT1, coding region is homologous to those for the phosphoribosyl anthranilate transferase from many microorganisms. This is due to a defect in tryptophan biosynthesis that leads to an accumulation of anthranilate, a fluorescent intermediate in the tryptophan pathway. PAT1 is a single-copy gene that complements all of the visible phenotypes of the different trp1 mutants. Experiments to determine the regulation of the PAT1 gene are in progress. The wild-type PAT1 promoter and several promoter deletions of PAT1 gene have been transformed into Arabidopsis tryptophan mutants. These constructs might identify promoter elements that control this patterns. We have isolated the homozygous lines in T3 seeds and analysed the protein levels using PAT antibody and PAT protein level increased two fold in pHSl07. Finally, the potential of using PAT1 as a selectable marker or visible reporter of gene expression is being explored.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.23
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• pp.10325-10328
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• 2015

RASSF1A, regarded as a candidate tumor suppressor, is frequently silenced and inactivated by methylation of its promoter region in many human tumors. However, the association between RASSF1A promoter methylation and lung cancer risk remains unclear. To provide a more reliable estimate we conducted a meta-analysis of cohort studies to evaluate the potential role of RASSF1A promoter methylation in lung carcinogenesis. Relevant studies were identified by searches of PubMed, Web of Science, ProQest and Medline databases using the following key words: 'lung cancer or lung neoplasm or lung carcinoma', 'RASSF1A methylation' or 'RASSF1A hypermethylation'. According to the selection standard, 15 articles were identified and analysised by STATA 12.0 software. Combined odds ratio (OR) and 95% confidence interval (CI) were used to assess the strength of the association between RASSF1A promoter methylation and lung cancer risk. A chi-square-based Q test and sensitivity analyses were performed to test between-study heterogeneity and the contributions of single studies to the final results, respectively. Funnel plots were carried out to evaluate publication bias. Overall, a significant relationship between RASSF1A promoter methylation and lung cancer risk (OR, 16.12; 95%CI, 11.40-22.81; p<0.001) with no between-study heterogeneity. In subgroup analyses, increased risk of RASSF1A methylation in cases than controls was found for the NSCLC group (OR, 13.66, 95%CI, 9.529-19.57) and in the SCLC group (OR, 314.85, 95%CI, 48.93-2026.2).