• YAKHAK HOEJI
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• v.39 no.2
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• pp.161-168
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• 1995

Brain dopamine systems play a central role in the control of movement, hormone release, and many complex behavior. The action of dopamine at its synapse is terminated predominately by high affinity reuptake into presynaptic terminals by dopamine transporter (DAT). The dopamine transporter(DAT) is membrane protein localized to dopamine-containing nerve terminals and closely related with cocaine abuse, Parkinsonism, and schizophrenia. In present study, the recombinant plasmid pRc/CMV-DAT, constructed by subcloning of a cDNA encoding a bovine DAT into eukaryotic expression vector pRc/CMV, was stably transfected into CV-1 cells(monkey kidney cell line). The DAT activities in the cell lines selected by Geneticin$^{R}$ were determined by measuring the uptake of $[^3H]$-dopamine. The transfected cell lines showed 30-50 fold higher activities than untransfected CV-1 cell line, and this result implies that DAT is well expressed and localized in transfected cells. The transfected cells accumulated $[^3H]$-dopamine in a dose-dependent manner with a $K_{m}$ of 991.6nM. Even though high doses of norepinephrine, epinephrine, serotonin, and choline neurotransmitters inhibited the uptake of $[^3H]$-dopamine, DAT in transfected cell line was proven to be much more specific to dopamine. The psychotropic drugs such as GBR12909, CFT, normifensine, clomipramine, desipramine, and imipramine inhibited significantly the dopamine uptake in tissue culture cells stably transfected with DAT cDNA. Radioactive in situ hybridization was done to map the cellular localization of DAT mRNA-containing cells in the adult rat central nervous system. The strong hybridization signals were detected only in the substantia nigra pars compacta and ventral tegmental area. The restricted anatomical localization of DAT mRNA-containing cells confirms the DAT as a presynaptic marker of dopamine-containing cells in the rat brain.

• Journal of Sericultural and Entomological Science
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• v.52 no.1
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• pp.39-44
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• 2014

We isolated highly-expressed genes in the posterior silk glands of silkworm on a previously study, which one of these was identified as RNA binding protein-1 homologue (RBP-1) gene. In this study, we investigated gene expressional characteristics of the RBP-1 depending on silkworm development stages and several tissues of the larvae, respectively. Northern blot hybridization analysis showed that the RBP-1 gene was expressed high in larval and pupal periods, and highly expressed than endogenous internal control gene (BmA3) on all tested larval tissues. In addition, we isolated and analyzed a phage DNA having 1,660 bp-long promoter region of the RBP-1 gene from a genomic DNA library. To study the RBP-1 gene promoter activity, RBP-1 (-740/+ 30) was amplified by PCR and subcloned into a pGL3 basic vector to generate pGL-RBP1. A luciferase report vector carrying RBP-1 gene promoter (770 bp) was tested by luciferase assay in Sf9 cells. In the result, the RBP-1 gene promoter was more efficient than constitutive promoter (BmA3) by approximately ten percent.

• Microbiology and Biotechnology Letters
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• v.44 no.4
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• pp.512-521
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• 2016

A bacterial strain was isolated from a soil sample collected on Jeju Island, Korea. The strain, designated WJ-2, exhibited a high xylanase activity, whereas cellulase activity was not detected. The 16S rRNA gene sequence of WJ-2 was highly similar to type strains of the genus Streptomyces. A neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showed that strain WJ-2 is phylogenetically related to Streptomyces atrovirens. Furthermore, DNA-DNA hybridization analysis confirmed that strain WJ-2 is a novel subspecies of Streptomyces atrovirens. The genomic DNA G+C content was 73.98 mol% and the major fatty acid present was anteiso-C15:0 (36.19%). The growth and xylanase production of strain WJ-2 were significantly enhanced by using soytone and xylan as nitrogen and carbon sources, respectively. Crude enzyme preparations from the culture broth of strain WJ-2 exhibited maximal total xylanase activities at pH 7.0 and $55^{\circ}C$. Thin-layer chromatography analysis revealed that the crude enzyme degrades beechwood xylan to yield xylobiose and xylotriose as the principal hydrolyzed end products.

• Microbiology and Biotechnology Letters
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• v.45 no.1
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• pp.43-50
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• 2017

A xylan-degrading bacterium (strain WJ-1) was isolated from soil collected from Jeju Island, Republic of Korea. Strain WJ-1 was characterized as a gram-positive, aerobic, and spore-forming bacterium. The predominant fatty acid in this bacterium was anteiso-$C_{15:0}$ (42.99%). A similarity search based on 16S rRNA gene sequences suggested that the strain belonged to the genus Streptomyces. Further, strain WJ-1 shared the highest sequence similarity with the type strains Streptomyces spinoveruucosus NBRC 14228, S. minutiscleroticus NBRC 13000, and S. glaucescens NBRC 12774. Together, they formed a coherent cluster in a phylogenetic tree based on the neighbor-joining algorithm. The DNA G+C content of strain WJ-1 was 74.7 mol%. The level of DNA-DNA relatedness between strain WJ-1 and the closest related species S. glaucescens NBRC 12774 was 85.7%. DNA-DNA hybridization, 16S rRNA gene sequence similarity, and the phenotypic and chemotaxonomic characteristics suggest that strain WJ-1 constitutes a novel subspecies of S. glaucescens. Thus, the strain was designated as S. glaucescens subsp. WJ-1 (Korean Agricultural Culture Collection [KACC] accession number 92086). Additionally, strain WJ-1 secreted thermostable endo-type xylanases that converted xylan to xylooligosaccharides such as xylotriose and xylotetraose. The enzymes exhibited optimal activity at pH 7.0 and $55^{\circ}C$.

• Korean Journal of Microbiology
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• v.33 no.1
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• pp.55-65
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• 1997

16S rRNA를 분석한 연구들은 자연 생태계에서 추출한 핵산을 이용하여 16rRNA 유전자의 염기서열을 분석하거나 특정 DNA probe를 이용한 hybridization 실험이 주류를 이루어 왔다. 특히 PCR 기법이 개발됨에 따라 적은 양의 시료를 대량으로 손쉽게 증폭시킬 수 있어 다양한 분야에 응용되고 있다. 세균 군집의 구조를 이해하는데 있어서 PCR 방법의 적용 대상은 주로 16S rRNA 유전자의 염기서열 해독분야이며 해양 생태계를 대상으로 많은 연구 결과가 보고되었다(11,13,21,26). 한편 자연 생태계의 개별적 미생물 분류룬들을 검출하기 위한 특정 oligonucleotide probe의 개발방법들은 미생물 군집의 유전적 다양성에 대한 정보 파악 이외에 배양이 어려운 혐기성 세균과 같은 특정 세균들의 동정에도 이용되고 있다(3,24,55). 본고에서는 세균 군집의 구조와 다양성을 연구하는데 적용 가능한 rRNA 분석방법들을 수계 생태계를 중심으로 살펴보고자 한다.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.12
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• pp.1837-1841
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• 2003

In an attempt to isolate novel molecules that may play a regulatory role in adipocyte differentiation, we devised an experimental strategy to identify adipose tissue-specific genes by modifying cDNA microarray technique. We used genefilter membranes containing approximately 15,000 rat non-redundant EST clones of which 4,000 EST were representative clones of known genes and 11,000 ESTs were uncharacterized clones. A series of hybridization of genefilter membranes with cDNA probes prepared from various rat tissues and nucleic acids sequence analysis allowed us to identify two adipose-tissue specific genes, adipocyte-specific secretory factor (ADSF) and H-rev107. Verification of tissue-specific expression patterns of these two genes by Northern blot analysis showed that ADSF mRNA is exclusive expressed in adipose tissue and the H-rev107 mRNA is predominantly expressed in adipose tissue. Further analysis of gene expression of ADSF and H-rev107 during 3T3-L1 adipocyte differentiation revealed that the ADSF and H-rev107 gene expression patterns are closely associated with the adipocyte differentiation program, indicating their possible role in the regulation of adipose tissue development. Overall, we demonstrated an application of modified cDNA microarray technique in molecular cloning, resulting in identification of two novel adipose tissue-specific genes. This technique will also be used as a useful tool in identifying novel genes expressed in a tissue-specific manner.

• Applied Biological Chemistry
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• v.39 no.5
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• pp.355-360
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• 1996

In vitro-tested ribozyme against synthesized cucumber mosaic virus (CMV) RNA (Agric. Chem. & Biotech. 37:56-63(1994)) was expressed in tobacco plant to develop virus resistant plants. The ribozyme sequence was linked to cauliflower mosaic virus 35S promoter and nopaline synthase(nos) terminator and this chimeric 35S-ribozyme-nos gene was sequenced. The sequenced chimeric gene was transferred to Agrobacterium tumefaciens LBA4404 using tri-parental mating system. The E. coli HB101 containing chimeric gene was incubated with E. coli HB101(pRK2073) as a helper and Agrobacterium tumefaciens LBA4404. Then Agrobacterium cells containing the ribozyme construct was cocultivated with tobacco leaf pieces. Ten different plants were regenerated from kanamycin containing MS medium. The presence of the ribozyme construct in the transgenic tobacco plants was confirmed by polymerase chain reaction (PCR). Seven different transgenic plants in ten different kanamycin resistant plants showed the expected size (570 base pairs) of 35S-ribozyme-nos gene fragment. Total RNAs were isolated from four different transgenic plants and separated on a 1% agarose gel containing formamide. Northern hybridization with 35S-ribozyme-nos gene fragment as a probe indicated that ribozyme transcripts may be degraded tv nuclease. Therefore, nuclease-resistant ribozymes are needed for the development of virus-resistant transgenic plants using ribozymes.

• Journal of Plant Biotechnology
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• v.3 no.3
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• pp.131-134
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• 2001

In $\lambda$-Zap II vector system, a cDNA library was constructed for the developing secondary xylem mRNA from hybrid poplar, Populus nigra x maximowiczii. A cDNA clone of 1.5 kb in size, pOMTB1.4 encoding a lignin-bispecific O-methyltransferase was screened by plaque hybridization using a probe of 540 bp cDNA amplified by polymerase chain reaction from the cDNA library and identified by nucleotide sequencing. Its nucleotide sequence contains one open reading frame of 366 amino acids. The deduced amino acid sequence in comparison with that of Populus tremuloides showed the differences of 9 amino acids and revealed 85-99% homology among alfalfa, poplar and aspen.

• Journal of the Korean Society of Tobacco Science
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• v.15 no.2
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• pp.161-166
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• 1993

A double - stranded cDNA fragment (436bp) encoding coat protein of tobacco mosaic virus(TMV) was derived from the total 480nucleotides gene after reverse transcription of TMV RNA, and subclorled into a plant expression vector pBl 121, resulting in pBL 430. The plasmid DNA containing this chimeric gene was moved from E. cofi to Agrobacterium tumefaciens strain A28l, and was introduced in시 the tobacco plant by the Agrobocterium Ti - mediated transformtion system. The transformants were selected on a selection media containing kanamycin. The shoots add roots could be differentiated from the explants and whole plants were obtained. From Southern blot hybridization analysis, DNA extracted from transformants, it could be conformed that the chimeric gene fragment was inserted into the genomic DNA of tobacco plant.

• Journal of Life Science
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• v.14 no.6 s.67
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• pp.1023-1027
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• 2004

The RAD3 gene of Saccharomyces cerevisiae is essential for the incision step of UV-induced excision repair. An yeast RAD3 gene has been previously isolated by functional complementation. In order to identify the RAD3 homologous gene from fungus Coprinus cinereus, we have constructed cosmid libraries from electrophoretically separated chromosomes of the C. cinereus. The 13 C. cinereus chromosomes were resolved by pulse-field gel electrophoresis, hybridized with S. cerevisiae RAD3 DNA, and then isolated RAD3 homologous DNA from C. cinereus chromosome. The RAD3 homolog DNA was contained in 3.2 kb DNA fragment. Here, we report the results of characterization of a fungus C. cinereus homolog to the yeast RAD3 gene. Southern blot analysis confirmed that the C. cinereus chromosome contains the RAD3 homolog gene and this gene exists as a single copy in C. cinereus genome. When total RNA isolated from the C. cinereus cells were hybridized with the 3.4 kb PvuII DNA fragment of the S. cerevisiae RAD3 gene, transcripts size of 2.8 kb were detected. In order to investigate whether the increase of the amount of transcripts by DNA damaging agent, transcript levels were examined after treating agents to the cells. The level of transcripts were not increased by untraviolet light (UV). This result indicated that the RAD3 homologous gene is not UV inducible gene. Gene deletion experiments indicate that the HRD3 gene is essential for viability of the cells and DNA repair function. These observations suggest an evolutionary conservation of other protein components with which HRD3 interacts in mediating its DNA repair and viability functions.