• Journal of Nutrition and Health
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• 제35권10호
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• pp.1053-1059
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• 2002

In post-genome period, the technique for identifying gene expression has been changed to high throughput screening. In the field of molecular nutrition, the need for this technique to clarify molecular function of the specific nutrient is essential. In this study, we have tested the zinc-regulated gene expression in zinc-deficient U937 cells, using cDNA microarray which is the cutting-edge technique to screen large numbers of gene expression simultaneously. The study result can be used for the preliminary gene screening data for clarifying, using monocyte U937 cell line, molecular Zn aspect in atherosclerosis. U937 cells were cultured in Zn-adequate (control, 12 $\mu$M Zn) or Zn-deficient (experimental, 0 $\mu$M Zn) ESMI media during 2 days, respectively. Cells were harvested and RNA was extracted. Total RNA was reverse-transcriptinized and synthesized cDNA probe labeled with Cy-3. fluorescent labeled cDNA probe was applied to microarray slide for hybridization slide, and after then, the slide was scanned using fluorescence scanner. ‘Highly expressed genes’ in Zn-deficient U937 cells, comparing to Zn-adequate group, are mainly about the genes for motility protein, immune system protein, oncogene and tumor suppressor and ‘Less highly expressed genes’ are about the genes for transcription, apoptosis associated protein, cell cycle, and several basic transcription factors. The results of this preliminary study imply the effectiveness of cDNA microarray for expression profiling of a singly nutrient deficiency, specially Zn. Furthur study, using tailored-cDNA array and capillary endothelial cell lines, would be beneficial to clarify molecular Zn function, more in detail.

• 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.

• Applied Biological Chemistry
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• 제40권1호
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• pp.30-33
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• 1997

환경친화형 생물농약개발을 위한 방안의 일환으로, 벼별구 등 농해충병원사상균 Metarhizium anisopliae의 분자생물학적 육종을 위해 영양요구성 돌연변이를 상보하는 선택유전자, ura5 (Orotate phosphoribosyl transferase)를 cloning하였다. Cloning방법으로는 기존에 알려진 사상균의 ura5 유전자들간에 확인된 상보성 염기배열을 합성하여, 이것을 primer로 사용하여 PCR기법에 의해 부분적으로 cloning하였다 또한, PCR기법에 의해cloning된 uras유전자단편의 염기배열을 결정한 결과, Trichoderma resei의 ura5유전자와는 아미노산수준에서 약 85%의 상동성을 나타내었으며, 이 단편을 이용하여 Metarhizium anisopliae의 genomic library로 부터 ura5유전자가 포함된 약 4.4 kb의 DNA단편을 cloning 하였다.

• 한국인지과학회:학술대회논문집
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• 한국인지과학회 2005년도 춘계학술대회
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• pp.177-181
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• 2005

기계학습에서 커널을 이용한 방법은 그 응용범위가 기계학습의 전반에 걸쳐 다양하게 이용되고 있으며, 그 성능 또한 기존의 방법들을 앞지르고 있다. 이는 기존의 비선형적 접근을 커널을 이용한 고차원 공간에서의 선형적 접근법으로 바꿈으로써 가능하게 되는 것이다. 다양한 분야에 적용되는 많은 커널들이 존재하며 각 커널들은 특별한 분야에 적용되기 쉽도록 다른 형태를 띠고 있기도 하지만, 커널로서 작용하기 위해 양한정 조건(positive definiteness)을 만족해야 한다. 본 연구에서는 DNA 문제에 직접 적용시킬 수 있는 방법으로서의 새로운 커널을 제시한다. 또한 매트로폴리스(Metropolis) 알고리즘을 이용하여 DNA의 hybridization과정을 모사함으로써 새로운 종류의 커널이 양한정(positive definite) 조건을 만족시킬 수 있는 방법을 제시한다. 새로 만들어진 커널이 행렬값을 형성해 나가는 과정을 살펴보면 인간이 예(instance)로부터 개념을 형성해 나가는 과정과 흡사한 양상을 보이는 것을 알 수 있다. 개념을 나타내는 좋은 예로서의 표본(prototype)으로부터 개념이 형성되어 가는 과정은 표본(prototype)이 아닌 예로부터 개념이 형성되는 과정과 다른 양상을 띠는 것과 같은 모양을 보인다.

• BMB Reports
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• 제38권1호
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• pp.28-33
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• 2005

A gene coding a novel isoform of carbamyl phosphate synthetase I (CPS1) was cloned from a human testicular library. As shown by cDNA microarray hybridization, this gene was expressed at a higher level in human adult testes than in fetal testes. The full length of its cDNA was 3831 bp, with a 3149 bp open reading frame, encoding a 1050-amino-acid protein. The cDNA sequence was deposited in the GenBank (AY317138). Sequence analysis showed that it was homologous to the human CPS1 gene. The putative protein contained functional domains composing the intact large subunit of carbamoyl phosphate synthetase, thus indicated it has the capability of arginine biosynthesis. A multiple tissue expression profile showed high expression of this gene in human testis, suggesting the novel alternative splicing form of CPS1 may be correlated with human spermatogenesis.

• Asian-Australasian Journal of Animal Sciences
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• 제16권12호
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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.

• BMB Reports
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• 제37권5호
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• pp.527-532
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• 2004

A subtracted cDNA library specific to osmotic stress of Haloxylon ammodendron (Mey.) Bge seedlings was constructed by suppression subtractive hybridization (SSH) and T/A cloning. SSH was performed between two groups of H. ammodendron seedlings, one was cultivated in Hoagland (H) solution as a driver and the other group was treated with osmotic stress of the Hoagland solution by the addition of 400 mM mannitol (M), as a tester. The library consisted of about 400 recombinant clones, with the average size being of 500 bp, ranging from 300 bp to 1500 bp. Using a PCR-select differential screening kit, 100 recombinant clones were randomly chosen from the subtracted cDNA library and hybridized with forward,reverse subtracted and unsubtracted probes for two rounds. As a result, 21 positive clones specific to osmotic stress were obtained and some of them were verified by Northern blot analysis. The sequencing analysis of 6 positive clones and the following homology comparison to GenBank [blastx] non-redundant databases characterized that two sequences obtained in this experiment may contribute to novel drought-related genes.

• Journal of Microbiology and Biotechnology
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• 제5권4호
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• pp.194-199
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• 1995

A portion (7.4 kb) of ribosomal DNA tandem repeat unit from a genome of the mushroom T. matsutake has been cloned. A 1.75 kb EcoRI fragment was cloned first using S. cerevisiae 255 rRNA gene as a probe, and this was then used for further cloning. A chromosomal walking experiment was carried out and the upstream region of the 1.75 kb fragment was cloned using SmaI/BamHI enzyme, the size was estimated to be 5.2 kb in length. Part of the downstream region of the 1.75 kb fragment was also cloned using XbaI/BamHI enzymes. Restriction enzyme maps of three cloned DNA fragments were constructed. Northern hybridization, using total RNA of T. matsutake, and the restriction fragments of three cloned DNAs as probes, revealed that all four ribosomal RNA genes (large subunit[LSU], small subunit [SSU], 5.85 and 5S rRNA genes) are present in the cloned region. The gene organization of the rDNA are regarded as an intergenic spacer [IGS]2 (partial) - SSU rRNA - internal transcribed spacer [ITS]1 - 5.8S rRNA - ITS2 - LSU rRNA - IGS1 -5S rRNA - IG52 (partial).

• Journal of Microbiology and Biotechnology
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• 제5권1호
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• pp.6-13
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• 1995

A gene coding for the $\beta$-galactosidase (lactase) of Kluyveromyces tragilis UCD 55-55 was isolated by complementation in Escherichia coli YMC9. From the plasmid library made from Sau3A-digested chromosomal DNA, one positive clone was selected. The cloned gene for $\beta$-galactosidase was on 7.3 kilobase pair DNA fragment, and a slightly low level of $\beta$-galactosidase enzyme activity was detecied in E. coli. It was also confirmed that the cloned gene comes from K. tragilis by DNA-DNA hybridization and immunochemical blotting experiments. In order to construct a new yeast strain having the metabolic ability for lactose, the cloned gene for K. tragilis $\beta$-galactosidase was inserted in yeast vector YEp24 and YRp17, and transformed into Saccharomyces cerevisiae YNN27 and Ml-2B. The yeast transformants showed the nearly the same $\beta$-galactosidase productivity as level of K. tragilis when uninduced, but these could not utilize lactose as a sole carbon source, presumably due to the lack of lactose transport system. Nevertheless, a slightly higher ethanol productivity was achieved by these transformants than S. cerevisiae or K. tragilis, in the medium containing glucose and lactose.

• 한국식물학회:학술대회논문집
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• 한국식물학회 1985년도 워크샵 및 심포지엄 북한산국립공원의 식생
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• pp.59-71
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• 1985

In rice, limited efforts have been made to identify genes by the use of insertional mutagens, especially heterologous transposons such as the maize Ac/Ds. We constructed Ac and gene trap Ds vectors and introduced them into the rice genome by Agrobacterium-mediated transformation. In this report, rice plants that contained single and simple insertions of T-DNA were analyzed in order to evaluate the gene-tagging efficiency. The 3'end of Ds was examined for putative splicing donor sites. As observed in maize, three splice donor sites were identified at the 3'end of the Ds in rice. Nearly 80% of Ds elements wered excised from the original T-DNA sites, when Ac cDNA was expressed under a CaMV 35S promoter. Repetitive ratoon culturing was performed to induce new transpositions of Ds in new plants derived from cuttings. About 30% of the plants carried at least one Ds that underwent secondary transposition in the later cultures. 8% of transposed Ds elements expressed GUS in various tissues of rice panicles. With cloned DNA adjacent to Ds, the genomic complexities of the insertion sites were examined by Southern hybridization. Half of the Ds insertion sites showed simple hybriodization patterns which could be easily utilized to locate the Ds. Our data demonstrate that the Ac/Ds mediated gene trap system could prove an excellent tool for the analysis of functions of genes in rice. We discuss genetic strategies that could be employed in a largee scale mutagenesis using a heterologous Ac/Ds family in rice.