• 한국양식학회지
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• 제10권1호
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• pp.33-37
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• 1997

미꾸라지를 대상으로 어류 유전자 이식을 위한 유용 model system으로 개발하기 위한 연구의 일환으로 잉어의$\beta$-actin 유전자의 promoter와 CAT 유전자가 융합되어 있는 외래 유전자 (pFV4CAT)를 미꾸라지에 이식, transgenic founder 미꾸라지를 생산하였다. PCR 분석결과, 이식된 외래 유전자는 부화 후 9개월된 성어에서 7.4-37.0%의 빈도로 존재하였으며 Southern blot 분석에 의해서 염색체 상의 삽입 가능성을 나타내었다. In situ immunohistochemical analysis 결과 이식된 외래 유전자는 미꾸라지 세포내에서 비교적 높은 빈도로 발현하였으며 그 발현 양상은 개체마다 다양하게 나타났다.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1997년도 춘계학술대회
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• pp.107-107
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• 1997

Cytochrome P450 enzymes have been intensively investigated in hepatic tissues and several mammalian cell lines. Compared to most studies about cytochrome P450 isozymes in liver in vivo and hepatic, cell lines in vitro, the study of cytochrome P450IA1 in human breast cancer cells could be very important to understand the mechanism of the regulation of CYPIA1 gene expression and cell growth. MCF-7 human breast cancer cells are well characterized to study estrogen and antiestrogen action due to the fact that they contain high level of estrogen receptor and have biological markers characterized. And also MCF-7 cells express high level of arylhydrocarbon hydroxylase activity and human cytochrome P450IA1 cDNA was cloned from MCF-7 cells. Ah receptor was characterized in many breast cancer cell lines and polycyclic aromatic hydrocarbon such as 3-MC induced the expression of CYPIA1 gene and cytochrome P450- dependent monooxygenase activity. We undertook a study to examine the effect of estrogens and other chemicals on the regulation of human CYPIA1 gene expression in MCF-7 cells via RTPCR analysis, that might help us to understand the mechanism of the regulation of CYPIA1 gene expression and MCF-7 cell growth. Expression vector containing the functional 5'-regulatory region of human CYPIA1 fused to the CAT reporter gene was transfected into estrogen receptor positive MCF-T cells or estrogen receptor negative MDA-MB-231 cells. After these cells were treated with various chemicals, RTPCR was carried out to measure both CYPIA1 mRNA and CAT mRNA levels. 1nM 3-MC increased in both P450 and CAT mRNA levels over those of control by two folds in MCF-7 cells but does not in MDA-MB-231 cells. Estrogen or tamoxifen or retinoic acid or chrysin decreased in both P450 and CAT mRNA levels that were induced by 3-MC in MCF-7 when each chemical was administered with 3-MC concomitantly. These results suggested that the level of CYPIA1 gene expression is modulated with estrogen-related molecules and make it possible to speculate that ER is related to CYPIA1 gene expression and cell growth in breast cancer cells. [Supported by grants from the Korean Ministry of Education ]

• 미생물학회지
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• 제27권4호
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• pp.317-322
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• 1989

A gene can be selectively overexpressed in E. coli by utilizing the phage T7 RNA polymerase's stringent recognition and active transcription of the T7 promoter. The T7 expression system was constructed such that the T7 RNA polymerase gene is under the control of lacUV5 promoter in one plasmid, and that the target gene, the promoterless chloramphenicol acetyltransferase (CAT) gene with E. coli ribosome binding site is under the control of T7 promoter in the other plasmid. Only the E. coli cells containing both plasmids show high resistance to chloramphenicol. When the copy number of the runaway plasmid containing the polymerase gene was varied by a temperature shift, amounts of the CAT protein synthesized upon induction was correspondingly changed as shown in SDS gel electrophoresis.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1995년도 제3회 추계심포지움
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• pp.153-155
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• 1995

To investigate the mechanism of the regulation of cytochrome P450IAl, the 5'-flanking region of a trout cytochrome P4501Al was cloned into the CAT basic expression vector at HindⅢ site. This trout Cytochrome P450IAl upstream DNA containing CAT construct was transfected into Hepa-1 cells .3MC treatment to hepa I cells transfected with trout P450IAl-CAT construct increased CAT protein and mRNA by 2.81 fold when it was compared with that of control. This increase CAT protein and mRNA was decreased by concomitantly treated flavonoids and aminopyrine. The level of CAT protein was 29.2-58.0% of 3MC stimulated CAT protein.

• 한국어류학회지
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• 제9권1호
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• pp.91-98
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• 1997

외래유전자, pFV4CAT이 이식된 transgenic 미꾸라지 계통 F1 및 F2를 대상으로 조직별 외래 유전자의 발현을 조사하였다. Transgenic F1에서 pFV4CAT의 mRNA 합성 여부를 조사하기 위해 정소(testis), 간(liver), 근육(muscle), 비장(spleen) 및 심장(heart) 조직을 RT-PCR로 분석한 결과, 조직별 mRNA 존재 여부는 F1 계통간 큰 차이를 나타내었으며, 다른 조직들에 비해 간(liver)과 비장(spleen)에서 보다 빈번히 발현하는 경향을 나타내었다. 외래 유전자에 의해 합성된 CAT 단백질을 ELISA로 정량화한 결과, 조직별 및 transgenic 계통별 다양한 차이가 있었으며, 다른 조직에 비해 근육과 심장에서 가장 높은 수준으로 발현하는 것으로 나타나 F1 한 계통의 근육에서, 대조군 수치의 최고 68배에 해당하는 CAT 발현이 관찰되였다. 반면 정소에서 가장 낮은 외래 유전자의 발현이 모든 transgenic line에서 관찰되었다.

• 미생물학회지
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• 제36권1호
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• pp.1-7
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• 2000

발광 박테리아인 Photobacterium 종들의 lux 오페론 하부 영역에서 riboflavin 생합성에 관여하는 유전자들(ribⅠ,Ⅱ,Ⅲ,Ⅳ)이 발견되었다. Photobacterium phosphoreum의 lux 유전자와 rib 유전자를 포함하는 intergenic 영역의 단일사슬 DNA가 P. phosphoreum의 mRNA에 의하여 S1 nuclease digestion에서 손상받지 않았으며, ribⅠ에 의하여 암호화되는 P. phosphoreum의 riboflavin synthase의 활성도가 lux-specific한 효소들인 luciferase 혹은 fatty acid reductase 활성도와 같이 bioluminescence intensity의 발현과 함께 대수기 말기에서 증가하는 박테리아 발광반응의 특이한 조절 체계인 'autoinduction' 양상을 보였다. 또한 P. leiognathi의 luxB로부터 ribⅡ까지 포함하는 DNA를 강력한 lux 프로모터와 reporter(chloramphenicol acetyl transferase, CAT) 유전자 사이에 삽입하고 접합(conjugation)의 방법으로 P. leiognathi에 유전자 전이(gene transfer)시켜 CAT reporter 유전자의 발현을 P. leiognathi에서 조사한 바, 그 유전자의 발현 정도에 큰 차이가 없었을 뿐만 아니라 이 구조에서 lux 프로모터를 제거하게 되면 CAT reporter 유전자의 발현이 전혀 나타나지 않았다. 이들 실험 결과들은 lux 유전자와 rib 유전자의 intergenic영역에 lux 오페론의 전사 종결 구조(transcriptional terminator)가 존재하지 않으며 ribflavin 생합성 유전자들이 그들 고유의 프로모터에 의하여 전사되는 것이 아니라 lux 오페론의 프로모터에 의하여 발현됨을 나타내는 것으로, 이는 Photobacterium 종들에서 lux 유전자와 rib 유전자들은 공동의 발현 조절 체계를 갖는 것으로 요약된다.

• Asian-Australasian Journal of Animal Sciences
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• 제19권9호
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• pp.1328-1334
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• 2006

This paper aimed to study the toxicity of arsanilic acid on rat primary hepatocytes in vitro by a modification of the perfusion method. The conditions included concentrations of 0, 1.085, 10.85, 108.5, 1,085 and 10,850 mg/kg arsanilic acid in RPMI 1,640 medium at rat hepatocytes plates respectively, each group had five repeats at $37^{\circ}C$ for 48 h. The rat primary hepatocytes survival ratio, DNA Ladder, activities of glutathione peroxidase (GSH-px), superoxide dismutase (SOD) and catalase (CAT) in hepatocytes, activity of SOD in the medium and the expression of gene bax in hepatocytes were measured at 12 h, 24 h and 48 h respectively. The results showed that arsanilic acid decreased the activities of GSH-px and SOD, and increased the activity of CAT in all dosages, and affected as positive DNA ladder. Although the SOD activities of both hepatocytes and medium in 1.085 mg/L arsanilic acid were significantly lower than the base line at 12 h, CAT activity in 10.85 mg/L arsanilic acid was significantly higher than the base line at 48 h, and all of the DNA ladders were positive, which means 1.085 mg/L arsanilic acid induced apoptosis at 24 h. The gene expression of bax was significantly upregulated in 1.085 mg/L arsanilic acid or higher for 24 h.The parameters in 1,085 mg/L and 10,850 mg/L arsanilic acid had more severe changes than the others at any time indicating that these levels of arsanilic acid were toxic hazards for hepatocyte survival. It was concluded that arsanilic acid induced a dosage- and time-dependent gene expression of bax, 1.085 mg/L arsanilic acid could be involved in rat liver cell apoptosis at 24 h. Arsanilic acid as additives in livestock feed could present potential toxic implications for farm animals.

• 미생물학회지
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• 제31권1호
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• pp.72-78
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• 1993

Human cytomegalovirus (HCMV) 의 immediate early(IE) 유전자의 promoter/enhancer 부위에 cAMP response element가 있다는 것으부터 caMP 가 HCMV 의 증식에 관여할 것이라고 생각할 수 있다. 이러한 가능성을 알아보기 위해 8-bromoadenosine 3', 5'-cyclic monophosphate (BrA) 와 papaverine 같은 세포내 cAMP 농도를 변화시키는 약제를 사용하여 HCMV 의 증식, DNA 합성 및 IE 유전자 발현에 대한 영향을 알아보았다. HCMV 증식과 DNA 합성은 papaverine 에 의해 억제된 반면, BrA 는 HCMV 의 증식에는 큰 영향을 주지 않았고 DNA 합성은 오히려 촉진시키는 것을 알 수 있었다. HCMV IE promoter 에 의해 작동되는 CAT 유전자를 함유한 plasmid pCMVIE/CAT 을 세포내로 transfection 시켰을 때, papaverine 을 처리한 세포에서는 CAT 효소 활성도가 감소한 반면 BrA 를 처리한 세포에서는 증가하였다. HCMV 에 감수성이 없는 HeLa 세포에서는 CAT 활성도가 감소성 세포인 HEL 세포에서보다 높게 나타난 반면, Vero 세포에서는 낮게 나타났다. 이들 비감수성 세포인 HEL 세포에서 보다 높게 나타난 반면 Vero 세포에서는 낮게 나타났다. 이 들 비감수성 세포에서의 CAT 활성도는 BrA 를 처리하여 주었을 때 모두 증가하였다. 이상과 같은 결과로부터 cAMP 는 HCMV 증식과 IE 유전자 발현에 어느 정도 관여한다는 것을 알 수 있다.

• Journal of Applied Biological Chemistry
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• 제43권1호
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• pp.1-4
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• 2000

Chloramphenicol acetyl CoA transferase (CAT) and angiotensin- converting enzyme inhibitory peptide (ACEI) were fused to C-terminal region of rice ubiquitin to examine the level of transcripts or enzyme activities in yeast. When two chimeric genes under an inducible Gall promoter control were transformed into Saccharomyces cerevisaie, both CAT and ACE inhibitory activities were enhanced by three to four-fold as compared to those containing no ubiquitin gene. However, the levels of transcripts of ubiquitin fused and un fused genes were not significantly different each other. Therefore, it was suggested that the expression of foreign genes was post-transcriptionally enhanced by fusion of plant ubiquitin in heterologous organisms such as yeast.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1996년도 춘계학술대회
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• pp.188-188
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• 1996

In order to gain insight into the mechanism of the regulation of cytochrome P450IAl by arylhydrocarbon, the 5'-flanking region of a trout CYP450IAl 5'flanking DNA was cloned into pCAT-basic vector and it was transfected into Hepa-1 cells. 3MC treatment to hepa Ⅰ cells transfected with fish CYP450IAl-CAT construct results in mRNA increased by 2.81 fold when it was compared with that of control This increase of mRNA was decreased by concomitantly treated flavonoids such as morin. The levels of CAT mRNA that was treated with morin was 29.2-58.0% of 3MC stimulated CAT mRNA. Further investigation to find out if there are DRE, XRE or negative regulatory cis element in CYP450IA1 gene was undertaken. Results of the deletion study of 5'flanking DNA of trout P450IA indicate the existance of the negative(-1600 ～ -1300). CAT mRNA was about two-fold higher in deleted trout CYP450IAl-CAT construct transfected cells compared to the wi Id type trout CYP450IAl-CAT construct transfected cells. And The stimulatory effect of 3MC was no longer observed in col Is containing deleted CAT construct. [Supported by grants from the Korean Ministry of Education]