• Journal of Microbiology
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• v.43 no.6
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• pp.523-528
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• 2005

Using the genomic library constructed at the downstream of the niiA promoter, which induces the over-expression of an inserted DNA fragment, we have attempted to screen the genes affecting growth or development by over-expression. The wild-type strain was transformed using the AMA-niiA(p) library and cultured on 1.2 M sorbitol media, in which asexual sporulation is induced, but sexual development is repressed. Over 100,000 strains transformed to $pyrG^+$ were analyzed with regard to any changes in phenotype. Consequently, seven strains were isolated for further analyses. These strains were designated NOT [niiA(p) over-expression transformants] stains. Four of the strains were of the inducible type, and the remaining strains were of the multi-copy suppression type. Two of the inducible-type strains, NOT 1 and NOT40, harbored genes which had been inserted in reverse direction, suggesting that the mutant phenotypes had been derived from an excess amount of anti-sense mRNA. Domain analyses of the deduced polypeptides from the DNA fragments rescued from the transformants revealed that NOT1, NOT40 and NOT6 harbored a LisH motif, a forkhead domain, and a $Zn(II)_2Cys_6$ binuclear zinc cluster, respectively.

• Journal of Ginseng Research
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• v.45 no.6
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• pp.754-762
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• 2021

Background: Ginsenoside Rh2, a major saponin derivative in ginseng extract, is recognized for its anti-cancer activities. Compared to coding genes, studies on long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) that are regulated by Rh2 in cancer cells, especially on competitive endogenous RNA (ceRNA) are sparse. Methods: LncRNAs whose promoter DNA methylation level was significantly altered by Rh2 were screened from methylation array data. The effect of STXBP5-AS1, miR-4425, and RNF217 on the proliferation and apoptosis of MCF-7 breast cancer cells was monitored in the presence of Rh2 after deregulating the corresponding gene. The ceRNA relationship between STXBP5-AS1 and miR-4425 was examined by measuring the luciferase activity of a recombinant luciferase/STXBP5-AS1 plasmid construct in the presence of mimic miR-4425. Results: Inhibition of STXBP5-AS1 decreased apoptosis but stimulated growth of the MCF-7 cells, suggesting tumor-suppressive activity of the lncRNA. MiR-4425 was identified to have a binding site on STXBP5-AS1 and proven to be downregulated by STXBP5-AS1 as well as by Rh2. In contrast to STXBP5-AS1, miR-4425 showed pro-proliferation activity by inducing a decrease in apoptosis but increased growth of the MCF-7 cells. MiR-4425 decreased luciferase activity from the luciferase/STXBP5-AS1 construct by 26%. Screening the target genes of miR-4425 and Rh2 revealed that Rh2, STXBP5-AS1, and miR-4425 consistently regulated tumor suppressor RNF217 at both the RNA and protein level. Conclusion: LncRNA STXBP5-AS1 is upregulated by Rh2 via promoter hypomethylation and acts as a ceRNA, sponging the oncogenic miR-4425. Therefore, Rh2 controls the STXBP5-AS1/miR-4425/RNF217 axis to suppress breast cancer cell growth.

• Journal of Veterinary Clinics
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• v.26 no.2
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• pp.123-129
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• 2009

Patent ductus arteriosus(PDA) is an abnormal shunt between the descending aorta and pulmonary artery through the incompletely closed ductus arteriosus and is the most common congenital heart defect in dogs. Recent human genetic studies found that a the gene mutation in transcription factor AP-2 beta(TFAP2B) was responsible for syndromic cases of PDA. Mutations in the TFAP2B gene are associated with certain congenital cardiac defects in humans that include PDA. In this study, we isolated the entire coding exons of canine TFAP2B gene for genetic screening in dogs with PDA. Analysis of the deduced amino acid sequence suggested that the canine TFAP2B are phylogenetically closer to the human TFAP2B(100% identity in amino acid sequence) than mouse and rat. In cTFAP2B gene screening, one single c.936+203G>A base change was found in affected Maltese dogs with PDA. However, further screening found the same base change in one unaffected control dog, suggesting this base change might be polymorphism. No other base changes were found in other dog breeds enrolled in this study. Because the base change was located in the intronic region and found in an unaffected control dog, TFAP2B might not be responsible for familial PDA in Malteses and sporadic cases of other dog breeds, although the gene promoter region should be investigated before reaching to this conclusion. A future study that may take this study further would be to collect more samples and to screen TFAP2B in various breeds of dogs with PDA and other various congenital heart defects.

• Korean Journal of Plant Tissue Culture
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• v.27 no.4
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• pp.259-268
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• 2000

Senescence is a sequence of biochemical and physiological events that lead to death of a cell, organ, or whole organism. Senescence is now clearly regarded as a genetically determined and evolutionarilly acquired developmental process comprising the final stage of development. However, in spite of the biological and practical importance, genetic mechanism of senescence has been very limited. Through forward and reverse genetic approaches, we are trying to reveal the molecular and genetic mechanism of senescence in plants, employing leaf organs of Arabidopsis as a model system. Using forward genetic approach, we have initially isolated several delayed senescence mutants either from T-DNA insertional lines or chemical-mutagenized lines. In the case of ore 4 and ore 9 mutants, the mutated genes were identified. The recent progress on characterization of mutants and identification of the mutated genes will be reported. We are also screening mutations from other various sources of mutant pools, such as activation tagging lines and promoter trap lines. Two dominant senescence-delayed mutants were isolated from the activation tagging pool. Cloning of the genes responsible for this phenotype is in progress. For reverse genetic approach, the genes that induced during leaf senescence were first isolated by differential screening method. We are currently using PCR-based suppression subtractive hybridization, designed to enrich a cDNA library for rare differentially expressed transcripts. Using this method, we have identified over 35 new sequences that are upregulated at leaf senescence stage. We are investigating the function of these novel genes by systemically generating antisense lines.

• Korean Journal of Medicinal Crop Science
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• v.15 no.2
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• pp.89-94
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• 2007

The effects of angiogenesis inhibitor from the extract libraries of Korean and Chinese medicinal plants were investigated using a protein microarray chip. Protein chip was constructed by immobilization of integrin ${\alpha}_5{\beta}_1$ on protein chip base plates and employed far screening active extracts that inhibit the integrin-fibronectin interaction from the extract libraries. The 100 extracts of medicinal plants were obtained from extract bank of National Institute of Crop Science, RDA. The 14 extracts among 100 extract libraries were shown efficient inhibition activity for the interaction between integrin-fibronectin. The medicinal plants of 14 extracts were Vitex negundo var. incisa (Lam.) C.B. Clarke, Epimedium koreanum Nakai, Cedrela sinensis A. Juss, Ipomea aquatica Forsk, Schisandra chinensis Baill, Pulsatilla koreana Nakai, Paeonia lactiflora Pall. var.hortensis Makino, Oenothera odorata, Allium chinense, Allium victorialis var. platyphyllum MAKINO, Polygonatum odoratum Druce var. pluriflorum Ohwi, Hosta lancifolia, Agrimonia pilosa L. var. japonica Nakai and Potentilla chinensis SER. The Paeonia lactiflora, Oenothera, and Agrimonia pilosa from these 14 extracts libraries were shown strong inhibition activity of integrin ${\alpha}_5{\beta}_1$.

• Food Science of Animal Resources
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• v.31 no.5
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• pp.658-662
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• 2011

In this study, 543 samples of press hams, sausages, processed ground meat and processed cheese acquired from retail markets in Seoul and Gyeonggi province in Korea from 2005 to 2010 were monitored using a one-step multiplex polymerase chain reaction (PCR) method that involves the amplification of specific soya or maize endogenous genes and the amplification of 35S promoter (p35S) and nopaline synthase terminator (tNOS) for GMO detection. Among the 543 samples, 477 samples were amplified for maize and/or soybean endogenous genes. Although one sausage sample collected in 2008 showed amplification of tNOS, the result was assumed to be false positive based on the results from further tests of other sausage samples of the same brand. Our results demonstrate the absence of GM soya and/or maze of livestock products in the Korean market during 2005-2010. In addition, the one-step multiplex PCR using previously constructed primer sets appears to be useful as a screening method for the detection of GMOs in processed livestock products. However, more specific methods should be established and employed to detect the event-specific GM gene for positive reaction samples by screening tests in processed livestock products.

• Korean Journal of Plant Tissue Culture
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• v.28 no.5
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• pp.231-237
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• 2001

A cDNA clone encoding metallothionein-like protein (CitMT45), which was reported by Moriguchi et al. (1998), was isolated from Citrus fruits cDNA library through differential screening. Our cDNA clone has longer 5'untranslated region, compared to it isolated by Moriguchi et al. (1998). RNA blot analysis showed that the mRNA was abundant in fleshes than peels, leaves, and flowers, as a single transcript. However, regardless of tissue types, the blots showed the similar expression patterns in the process of development with some different profile. These results suggest that CitMT45 may play important roles in the development and/or senescence of various tissues of Citrus. A genomic clone corresponding to CitMT45 was isolated and found to have three exons and two introns. A primer extension analysis suggested that the transcription of CitMT45 gene was started at three start sites with different degrees. The 5'-flanking region was shown to contain a putative metal regulatory element (MRE) and low- temperature responsive element which suggests the possibility of metal-and cold-regulated transcription, respectively.

• Microbiology and Biotechnology Letters
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• v.35 no.4
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• pp.272-277
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• 2007

The physiological responses of microorganisms to specific nutrient limitation can be regulated at the transcriptional levels. In this study, in order to develop the Pichia pastoris-derived promoter inducible by nutrient-limited condition, we constructed cDNA libraries using RT-PCR of total RNA from P. pastoris in steady-states of phosphate-limited chemostat with different dilution rates. Various genes were detected from cDNA library. Among these genes, the gene encoding putative sodium/phosphate ($Na^+$/Pi) symporter (NPS), high affinity transporter of phosphate, was detected. It was observed that expression of NPS increased in a manner specific to phosphate-limited condition through Northern blot. Therefore, it is thought that the promoter from NPS gene may have the potential as auto-inducible promoter by phosphate-limited culture condition without inducer.

• BMB Reports
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• v.44 no.9
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• pp.607-612
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• 2011

Several investigators have shown that CpG-DNA has outstanding effects as a Th1-responsive adjuvant and that its potent adjuvant effects are enhanced by encapsulation with a liposome of proper composition. In this study, we showed that encapsulation with phosphatidyl-${\beta}$-oleoyl-${\gamma}$-palmitoyl ethanolamine (DOPE): cholesterol hemisuccinate (CHEMS) complex enhances the immunostimulatory activity of CpG DNA and the binding of CpG-DNA to TLR9. We also examined involvement of myeloid differentiation protein (MyD88) and NF-${\kappa}B$ activation in liposome-encapsulated CpG-DNA-induced IL-8 promoter activation. In this manuscript, the natural phosphodiester bond CpG-DNA encapsulated by DOPE : CHEMS complex is designated as Lipoplex(O). Importantly, we successfully screened B cell epitopes of envelope protein (E protein) of hepatitis C virus (HCV-E) and attachment glycoprotein G of human respiratory syncytial virus (HRSV-G) by immunization with complexes of several peptides and Lipoplex(O) without carriers. Therefore, Lipoplex(O) is potentially applicable as a universal adjuvant for peptide-based epitope screening and antibody production.

• The Journal of Korean Society of Virology
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• v.28 no.1
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• pp.21-30
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• 1998

Synthetic genes encoding the gag p24 and the part of the envelope protein gp41 of the human immunodeficiency virus (HIV-1) were cloned and overexpressed as fusion proteins in Escherichia coli, using an expression vector carrying T7 promoter and the poly-histidine leader, sequence. The overexpressed p24 fusion protein was purified by centrifugation, Ni-affinity chromatography and CM-sepharose chromatography. The overexpressed gp41 fusion protein was purified by centrifugation, $C_4$ chromatography and DEAE-sepharose chromatography. The purified fusion proteins showed a high level of purity and immunoreactivity in SDS-polyacrylamide gel electrophoresis and western blot analysis. These results suggest that this prokaryotic - expression purification method is suitable for obtaining a large amount of the viral antigen which may be useful for screening of antibodies to HIV-1 in human blood samples.