• Proceedings of the Botanical Society of Korea Conference
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• 1999.07a
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• pp.21-35
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• 1999

We have previously isolated OsMADS4 gene that is a member of the class B MADS box genes from rice. In this study, another member of the class B MADS box genes was isolated from rice flower by the yeast two-hybrid screening method using OsMADS4 as bait. RNA blot analyses revealed that the clone, OsMADS16, was expressed in the second and third whorls, whereas the OsMADS4 transcripts were present in the second, third, and fourth whorls. These expression patterns of the OsMADS16 and OsMADS4 genes are very similar with those of AP3 and PI, the class B genes of Arabidopsis, respectively. In the yeast two-hybrid system, OsMADS4 interacted only with OsMADS16 among several rice MADS genes investigated, suggesting that OsMADS4 and OsMADS16 function as a heterodimer in specifying sepal and petal identities. We have also isolated OsMADS6 gene using OsMADS1 as a probe. Both are members of the AGL2 MADS family. Various MADS genes that encode for protein-protein interaction partners of the OsMADS6 protein were isolated by the yeast two-hybrid screening method. A majority of these genes belong to the AGL2 family. Sequence Homology, expression pattern, and ectopic expression phenotypes indicated that one of the interaction partners, OsMADS14, appears to be homologous to API, the class A MADS gene of Arabidopsis.

• BMB Reports
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• v.43 no.8
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• pp.567-572
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• 2010

In this study, we cloned the ERF-B3 subfamily transcription factor gene BnaERF-B3-hy15 from Brassica napus L. Huyou15. This 600 bp gene encodes a 199 amino acid classic ethylene responsive factor (ERF), which shown no binding or very weak binding GCC box-binding activity by the yeast one-hybrid assay. We used gene shuffling and the yeast one-hybrid system to obtain three mutated sequences that can bind to the GCC box. Sequence analysis indicated that two residues, Gly156 in the AP2 domain and Phe62 at the N-terminal domain were mutated to arginine and serine, respectively. Changes of Gly156 to arginine and Phe62 to serine increased the GCC-binding activity of BnaERF-B3-hy15 and the alter of Gly156 to arginine changed the AP2-domain structure of BnaERF-B3-hy15.

• Journal of Korean Society of Water and Wastewater
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• v.19 no.1
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• pp.98-105
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• 2005

The effects of chlorination on the elimination of three estrogenic chemicals such as $17{\beta}$-estradiol (E2), nonylphenol (NP) and bis-phenol A (BPA) were investigated using yeast two-hybrid assay (YTA), estrogen receptor competition assay (ER-CA), and high-performance liquid chromatography/mass spectrometer (LC/MS). Results of YTA, ECA and the analysis of LC/MS indicated that the estrogenic activity of above mentioned three endocrine disruptors were significantly reduced as the result of chlorination. The decrease in estrogenic activity paralleled with decrease in estrogenic chemicals under the influence of free chlorine. One common characteristic of estrogenic chemicals is the presence of a phenolic ring. Considering that a phenolic ring is likely to undergo some sort of transformation in aqueous chlorination solution, the above mentioned results may be applied to the rest of the other estrogenic chemicals in natural waters.

• Biomolecules & Therapeutics
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• v.17 no.1
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• pp.12-16
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• 2009

One of the WHSC1/MMSET/NSD2 variant RE-IIBP is a histone H3-K27 methyltransferase with transcriptional repression activity. Overexpression of RE-IIBP in various types of leukemia suggests it's role in leukemogenesis. Here we identify two proteins, KRAB zinc finger protein ZNF 350 and enolase-1 as RE-IIBP interacting proteins by yeast two-hybrid screening and confirmed direct interaction in vivo and in vitro. Both proteins have been known for their role in transcriptional repression. Reporter assays using transient transfection demonstrated that both ZNF 350 and enolase-1 proteins synergistically repressed transcription with RE-IIBP, respectively. These results indicate both proteins have roles in RE-IIBP mediated transcriptional repression by involving co-repressor complex.

• Journal of Microbiology
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• v.34 no.4
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• pp.327-333
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• 1996

Yeast, like many other microbes, encounters large variations in ambient pH in their natural environments. Microorganisms capable of growing over a wide pH range require a versatile, efficient pH homeostatic mechanism protecting intracellular processes against extremes of pH. In several organisms, fusions to the bacterial lacZ gene have been extremely useful for the identification of genes expressed at different time during the life cycle or under different growth conditions. In this study, using the lacZ gene screening system, we surveyed a large number of yeast strains with lacZ insertion to identify genes regulated by pH. A yeast genomic library was constructed and inserted with lacZ by a shuttle mutagenesis procedure. The yeast transformants were individually picked up with a toothpick, replica-plated, and grown in alkaline pH medium. Among the 35,000 colonies screened, 10 candidate strains were identified initially by the $\beta$-gal assay. We finally confirmed two yeast strains carrying the genes whose expression are strictly dependent on pH of growth medium. One of the fusions showing a 10-fold induction in expression level in response to alkali pH was selected and further characterized. The pH-regulated gene was cloned by inverse PCR and a partial sequence of the gene was determined. Identification and characterization of the gene is currently under investigation.

• Journal of Microbiology and Biotechnology
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• v.12 no.2
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• pp.286-291
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• 2002

Histone deacetylase I (HDAC1) works as one of the components in a nucleosome remodeling (NuRD) complex that consists of several proteins, including metastasis-associated protein 1 (MTA1). Since the protein-protein interaction of HDAC1 and MTA1 would appear to be important for both the integrity and functionality of the HDAC1 complex, the interruption of the HDAC1 and MTA1 interaction may be an efficient way to regulate the biological function of the HDAC1 complex. Based on this idea, a yeast two-hybrid system was constructed with HDAC1 and MTA1 expressing vectors in the DNA binding and activation domains, respectively. To verify the efficiency of the assay system, 3,500 microbial metabolite libraries were tested using the paper disc method, and KB0699 was found to inhibit the HDAC1 and MTA1 interaction without any toxicity to the wild-type yeast. Furthermore, KB0699 blocked the interaction of HDAC1 and MTA1 in an in vitro GST pull down assay and induced morphological changes in B16/BL6 melanoma cells, indicating the interruption of the HDAC1 complex function. Accordingly, these results demonstrated that the yeast assay strain developed in this study could be a valuable tool for the isolation of a HDAC1 complex disruptor.

• Animal cells and systems
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• v.6 no.4
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• pp.317-322
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• 2002

To improve conventional yeast one-hybrid screening, we have developed an efficient mammalian one-hybrid system that allows rapid isolation of com-plementary DNAs which are able to induce human p14$^{ARF}$. tumor suppressor gene. A 1.5 kb promoter region of p14$^{ARF}$ was fused to EGFP to generate ARF promoter-EGFP reporter vector. This reporter plasmid was stably trans-fected into NIH3T3 cells for generation of reporter cell line. When the reporter cell line was infected with E2F-1 together with excess amounts of empty vector, the cells that received the positive modulator were readily identifiable by green fluorescence using FACS. The GFP-positive cells were cloned directly from the cultured cells and expanded in bulk culture. The genomic DNAs from GFP-positive cells were prepared and the CDNA insert in integrated retroviral genome was recovered by PCR using primers annealing to the retroviral vector sequences flanking the insert-cloning site. This system should be useful for efficient screening of expression CDNA libraries in mammalian cells to identify novel upstream regulators for spe-cific genes by one-hybrid interaction.ion.

• Journal of Ginseng Research
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• v.41 no.3
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• pp.403-410
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• 2017

Background: Prenyltransferases catalyze the sequential addition of isopentenyl diphosphate units to allylic prenyl diphosphate acceptors and are classified as either trans-prenyltransferases (TPTs) or cis-prenyltransferases (CPTs). The functions of CPTs have been well characterized in bacteria, yeast, and mammals compared to plants. The characterization of CPTs also has been less studied than TPTs. In the present study, molecular cloning and functional characterization of a CPT from a medicinal plant, Panax ginseng Mayer were addressed. Methods: Gene expression patterns of PgCPT1 were analyzed by quantitative reverse transcription polymerase chain reaction. In planta transformation was generated by floral dipping using Agrobacterium tumefaciens. Yeast transformation was performed by lithium acetate and heat-shock for $rer2{\Delta}$ complementation and yeast-two-hybrid assay. Results: The ginseng genome contains at least one family of three putative CPT genes. PgCPT1 is expressed in all organs, but more predominantly in the leaves. Overexpression of PgCPT1 did not show any plant growth defect, and its protein can complement yeast mutant $rer2{\Delta}$ via possible protein-protein interaction with PgCPTL2. Conclusion: Partial complementation of the yeast dolichol biosynthesis mutant $rer2{\Delta}$ suggested that PgCPT1 is involved in dolichol biosynthesis. Direct protein interaction between PgCPT1 and a human Nogo-B receptor homolog suggests that PgCPT1 requires an accessory component for proper function.

• BMB Reports
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• v.38 no.5
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• pp.602-608
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• 2005

As a crucial transcription factor family, heat-shock factors were mainly analyzed and characterized in tomato and Arabidopsis. In this study, we isolated two putative heat shock factors OsHSF6 and OsHSF12 that interact specifically with heat-shock element (HSE) from Oryza sativa L by yeast one-hybrid method. The full-length cDNA of OsHSF6 and OsHSF12 have 1074bp and 920bp open reading frame (ORF), respectively. Analysis of the deduced amino acid sequences revealed that OsHSF6 was a class A heat shock factor (HSF) with all the conserved sequence elements characteristic of heat stress transcription factor, while OsHSF12 was a class B HSF with C-terminal domain (CTD) lacking of AHA motif. Bioinformatic analysis showed that the sequences and structures of two HSFs' DNA binding domain (DBD) had a high similarity with LpHSF24. The results of RT-PCR indicated OsHSF6 gene was expressed immediately after rice plants exposure to heat stress, and the transcription of OsHSF6 gene accumulated primarily in immature seeds, roots and leaves. However, we did not find the transcription of OsHSF12 gene in different organs and growth periods. Our results implied that OsHSF6 might be function as a HSF regulating early expression of stress genes in response to heat shock, and OsHSF12 might be act as a synergistic factor to regulate the expression of down-stream genes.

• BMB Reports
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• v.38 no.4
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• pp.440-446
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• 2005

A cDNA that was rapidly induced upon abscisic acid, cold, drought, mechanical wounding and to a lesser extent, by high salinity treatment, was isolated from Arabidopsis seedlings. It was classified as DREB subfamily member based on multiple sequence alignment and phylogenetic characterization. Since it encoded a protein with a typical ERF/AP2 DNA-binding domain and was closely related to the TINY gene, we named it TINY2. Gel retardation assay revealed that TINY2 was able to form a specific complex with the previously characterized DRE element while showed only residual affinity to the GCC box. When fused to the GAL4 DNA-binding domain, either full-length or its C-terminus functioned effectively as a trans-activator in the yeast one-hybrid assay while its N-terminus was completely inactive. Our data indicate that TINY2 could be a new member of the AP2/EREBP transcription factor family involved in activation of down-stream genes in response to environmental stress.