• 미생물학회지
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• 제5권2호
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• pp.61-68
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• 1967

In the process of the incorporation of orthophsphate into protein and other cell constituents, the role of inorganic polyphosphate and RNA-polyphosphate complex and the correlation between them were pursued by analyzing the contents of $^{32}P$ and total P in various fractions of Chlorella cells, which had been uniformly labeled with $^{32}P$ before the inoculation in a normal "cold" medium or P-free medium during the culture. The effects of ionizing radiation and various micronutritional-element deficiencies on the phosphate incorporation into, and biosynthesis of, protein and other introgenus compounds in the cells were also observed. When the uniformly $^{32}P$-labeled algae were grown in a normal "cold" medium the contents of $^{32}$ P in the fractions of protein, DNA and RNA-polyphosphate complex increased, but those in the fraction of acid-insoluble polyphosphate decreased. On the other hand, amount of $^{32}P$in the fraction of RNA was almost unchanged in spite of rapid increase of the total P. In the growing period of $^{32}P$-labeled algae in a P-free medium, amounts of $^{32}P$ in the fractions of DNA, protein and lipid increased, while those in the fractions of RNA-polyphosphate and inorganic polyphosphates decreased. When the algal cells were irradiated with about 70, 000r of gamma-rays before the inoculation in the medium, amounts of phosphate in the fractions of DNA, RNA, nucleotides and protein decreased during the culture, compared with those of the control. However, the phosphate content in the fraction of acid-insoluble polyphosphate of the irradiated cells increased than those of the control. In the growing period of the algae in a Mo-free, medium, amounts of acid-soluble total phosphate and nucleotides of the cells increased, while the amounts of residual protein and RNA decresed compared with those of the normal cells. Amounts of alkali-labile protein and phospholipid of the cells grown in a B-free medium decreased, whereas amount of phosphate in acid-soluble fraction increased compared with the control. In general, the contents of protein and RNA in each microelement deficient cells decreased more or less, compared with those in the normal cells.in the normal cells.

• Fisheries and Aquatic Sciences
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• 제3권1호
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• pp.64-70
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• 2000

DNA probes for the l6S rRNA have been designed for the detection of Edwardsiella tarda. In order to accomplish this purpose, the l6S rRNA gene from E. tarda has been cloned and sequenced. Two highly feasible oligonucleotide probe sites have been determined by the database analysis programs presented by PCGENE and BLAST. These two probes have been evaluated by slot blot hybridization analysis. Hetero- and homo-trimeric templates have been synthesized using these two probe sites. The templates have been further multimerized by PCR to generate between 150 and 300 bp long DIG-11-dUTP labeled probes. Unlike 3' end labeled oligonucleotide probes or templates, multimerized probes showed no cross­hybridization in the given experimental condition. Furthermore, a significant increase in sensitivity has been observed with these probes. This method, we presented here, may be useful for the designing of probes for the detection of other fish pathogenic microorganisms also.

• 미생물학회지
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• 제47권2호
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• pp.158-162
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• 2011

Proteus mirabilis 전사 조절($\underline{P}$roteus $\underline{m}$irabilis $\underline{t}$ranscription $\underline{r}$egulator ) 단백질의 중금속 결합 부위에 대한 아미노산 서열분석에서 PMTR 단백질은 ZntR (아연 저항성) 단백질이 아닌 CueR (구리 저항성) 단백질과 동일한 환경이다. 그리고 겔시프트 법(gel shift assay) 실험에 의하면 PMTR 단백질은 Escherichia coli의 zntA (zinc-translocating P-type ATPase gene) 프로모터에 결합하지 않고 copA (copper-translocating P-type ATPase gene) 프로모터와 Proteus mirabilis에서 atpase (copper-translocating P-type ATPase gene) 프로모터에 결합하였다. DNase I protection 실험에서 PMTR 단백질 결합부위와 DNase I 민감성 염기들이 관찰되었다. P. mirabilis atpase 프로모터에서 민감성 염기로 주형가닥(template strand)에서 C와 A 그리고 비주형가닥(non-template strand)에서 G와 C 염기들이다. 이런 민감성 염기들은 다른 MerR 패밀리 단백질에서 또한 관찰되었으며, 이것은 단백질에 의한 DNA bending을 의미한다.

• Preventive Nutrition and Food Science
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• 제8권4호
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• pp.390-394
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• 2003

In the post-genome period, the technique for identifying gene expression has been progressed to high throughput screening. In the field of molecular nutrition, the use of screening techniques to clarify molecular function of specific nutrients would be very advantageous. In this study, we have evaluated Zn-regulated gene expression in Zn-deficient, homocystein-treated EA.hy926 cells, using cDNA microarray, which can be used to screen the expression of many genes simultaneously. The information obtained can be used for preliminary assessment of molecular and signaling events modulated by Zn under pro-atherogenic conditions. EA.hy926 cells derived from human umbilical vein endothelial cells were cultured in Zn-adequate (control, 15 $\mu$M Zn) or Zn-deficient (experimental, 0 $\mu$M Zn) Dulbecco's MEM media under high homocysteine level (100 $\mu$M) for 3 days of post-confluency. Cells were harvested and RNA was extracted. Total RNA was reverse-transcribed and the synthesized cDNA was labeled with Cy3 or Cy5. Fluorescent labeled cDNA probe was applied to microarray slides for hybridization, and the slide was then scanned using a fluorescence scanner. The expression of seven genes was found to be significantly decreased, and one significantly increased, in response to treatment of EA.hy926 cells with Zn-deficient medium, compared with Zn-supplemented medium. The upregulated genes were oncogenes and tumor suppressor genes, cell cycle-related genes and transporter genes. The down-regulated gene was RelB, a component of the NF-kappaB complex of transcription factors. The results of this study imply the effectiveness of cDNA microarray for expression profiling of a singly nutrient deficiency, namely Zn. Furthur study, using tailored-cDNA array and vascular endothelial cell lines, would be beneficial to clarify the molecular function of Zn in atherosclerosis, more in detail.

• 약학회지
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• 제40권4호
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• pp.442-448
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• 1996

Mitomycin C(MMC) has been used as an anticancer drug and behaves as an alkylating agent forming covalent cross-link between complementary strands of double strand DNA. The purpose of this research was to determine number of DNA adducts, formed in vivo by Mitomycin C, in mouse organs. DNAs from liver, lung, brain and pancreas were isolated and used for $^{32}P$-postlabelling. The labeled nucleotides were separated by 2D-TLC and subjected to autoradiography. Numbers of MMC-DNA adducts were 9,9,5,4 in liver, pancreas, lung and brain, respectively.

• 방사선산업학회지
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• 제5권3호
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• pp.243-247
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• 2011

In this study, a convenient method for the selective immobilization of single strand DNA (ssDNA) on a polymer surface was described. A positively charged polyelectrolyte, poly(diallyldimethylammonium chloride) (PDDA), was spin-coated on a tissue culture petridish and the micropatterns of the PDDA were formed by selective ion implantation through a pattern mask. The surface property of the implanted PDDA was investigated by using a surface profiler and FT-IR spectrometer. Cy3-labeled ssDNA was selectively immobilized on the PDDA patterns through ionic interaction and thus, well-defined ssDNA patterns were obtained.

• Journal of Microbiology and Biotechnology
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• 제14권6호
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• pp.1239-1248
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• 2004

The methylotrophic yeast Hansenula polymorpha has been extensively studied as a model organism for methanol metabolism and peroxisome biogenesis. Recently, this yeast has also attracted attention as a promising host organism for recombinant protein production. Here, we describe the fabrication and evaluation of a DNA chip spotted with 382 open reading frames (ORFs) of H. polymorpha. Each ORF was PCR-amplified using gene-specific primer sets, of which the forward primers had 5'-aminolink. The PCR products were printed in duplicate onto the aldehyde-coated slide glasses to link only the coding strands to the surface of the slide via covalent coupling between amine and aldehyde groups. With the partial genome DNA chip, we compared efficiency of direct and indirect cDNA target labeling methods, and found that the indirect method, using fluorescent-labeled dendrimers, generated a higher hybridization signal-to-noise ratio than the direct method, using cDNA targets labeled by incorporation of fluorescence-labeled nucIeotides during reverse transcription. In addition, to assess the quality of this DNA chip, we analyzed the expression profiles of H. polymorpha cells grown on different carbon sources, such as glucose and methanol, and also those of cells treated with the superoxide­generating drug, menadione. The profiles obtained showed a high-level induction of a set of ORFs involved in methanol metabolism and oxidative stress response in the presence of methanol and menadione, respectively. The results demonstrate the sensitivity and reliability of our arrays to analyze global gene expression changes of H. polymorpha under defined environmental conditions.

• Journal of Photoscience
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• 제9권2호
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• pp.488-490
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• 2002

DNA photodamage mediated by photosensitizers are believed to play an important role in solar UVA carcinogenesis. We investigated the relationship between the DNA-damaging abilities of photoexcited xanthone analogues (as photosensitizers) and their highest occupied molecular orbital (HOMO) energies. DNA damage was examined using /sup 32/P-labeled DNA fragments obtained from the p53 tumor suppressor gene. These compounds induced DNA photodamage in a similar manner, and the extents of DNA damage were following order: xanthone> thioxanthone > acridone. Photoexcited xanthone caused nucleobase oxidation specifically at 5'-G of GG sequence in double-stranded DNA. An oxidative product of 2'-deoxyguanosine, 8-hydroxy-2'-deoxyguanosine (8-OHdG), was detected, and the amount was decreased by DNA denaturation. These findings suggest that photoexcited xanthone generates 8-OHdG at 5'-G of GG in double-stranded DNA through electron transfer. The calculated HOMO energies of these photosensitizers decreased in the following order: xanthone> thioxanthone > acridone. This study has demonstrated that DNA-damaging abilities of these photosensitizers increased exponentially with an increase in their HOMO energies.

• BMB Reports
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• 제33권5호
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• pp.417-421
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• 2000

Mitochondrial DNA (mtDNA) mutation was investigated in a hepatoma patient using a polymerase chain reaction (PCR) and an in situ hybridization technique. Biotin-labeled probes for the subunit m of cytochrome c oxidase revealed differences in the in situ hybridization. A PCR assay using biopsied and microdissected tissues showed that common deletion (4,977 bp) was more pronounced in the cancer region than in the normal parts of the same patient. These results suggest that mtDNA deletion might be associated with tumorigenesis in hepatoma.

• Archives of Pharmacal Research
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• 제28권1호
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• pp.93-99
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• 2005

Intracellular trafficking of transferrin-conjugated dimethyldioctadecyl-ammonium bromide liposome $(T_f-liposome)/DNA$ complexes in HeLa cells was studied using the double-labeled fluorescence technique and confocal microscopy. The size of the $T_f-liposome/DNA$ complex was about 367 nm in diameter and the zeta-potential of it at a 5:1 (w/w) ratio was almost neutral. The intracellular pathway of the $T_f-liposome/DNA$ complex, noted as green (FITC), red (rhodamine) or yellow (FITC + rhodamine) fluorescence, was elucidated from the plasma membrane to the endosome (or lysosome), and finally to the nucleus. The results of this study indicate that plasmid DNA enters into the nucleus not only as a free form but as an associated form complexed with $T_f-liposome$. More interestingly, the $T_f-liposome$ undergoes a nuclear location in the form of ordered structures. This could be a very useful piece of information in designing a safe and advanced gene delivery system.