본 학위논문의 목적은 바이러스 유전자의 기능을 밝히기 위한 준비 단계로 박테리아 유래 테트라싸이클린 repressor와 테트라싸이클린(tetracycline)을 이용하여 바이러스 유전자의 기능을 밝히기 위한 전사 억제 및 전사 활성시스템 개발에 있다. 본 논문은 4부분으로 구성되어 있는데 제 I은 서론 부분으로 본 논문의 이해를 돕기 위하여 테트라싸이클린 및 테트라싸이클린 repressor, 논문의 주 모델 시스템인 human cytomegal...
본 학위논문의 목적은 바이러스 유전자의 기능을 밝히기 위한 준비 단계로 박테리아 유래 테트라싸이클린 repressor와 테트라싸이클린(tetracycline)을 이용하여 바이러스 유전자의 기능을 밝히기 위한 전사 억제 및 전사 활성시스템 개발에 있다. 본 논문은 4부분으로 구성되어 있는데 제 I은 서론 부분으로 본 논문의 이해를 돕기 위하여 테트라싸이클린 및 테트라싸이클린 repressor, 논문의 주 모델 시스템인 human cytomegalovirus (HCMV), 진핵세포에서의 전사개시 기작에 대한 일반적인 내용을 다루었다. 제 II, III, IV, V는 tTA에 의한 전사억제, 전사활성시스템, 전사활성시스템에서 tTA/TetR이종 dimer의 효과 연구에 대한 재료 및 방법, 결과, 고찰, 결론에 대해 각각 다루었다.
본 학위논문의 목적은 바이러스 유전자의 기능을 밝히기 위한 준비 단계로 박테리아 유래 테트라싸이클린 repressor와 테트라싸이클린(tetracycline)을 이용하여 바이러스 유전자의 기능을 밝히기 위한 전사 억제 및 전사 활성시스템 개발에 있다. 본 논문은 4부분으로 구성되어 있는데 제 I은 서론 부분으로 본 논문의 이해를 돕기 위하여 테트라싸이클린 및 테트라싸이클린 repressor, 논문의 주 모델 시스템인 human cytomegalovirus (HCMV), 진핵세포에서의 전사개시 기작에 대한 일반적인 내용을 다루었다. 제 II, III, IV, V는 tTA에 의한 전사억제, 전사활성시스템, 전사활성시스템에서 tTA/TetR이종 dimer의 효과 연구에 대한 재료 및 방법, 결과, 고찰, 결론에 대해 각각 다루었다.
The tetracycline repressor (TetR) from Escherichia coli was used to develop a system in which expression of herpesviral genes could be regulated. To develop the TetR-mediated gene repression system in HCMV, recombinant virues containing the US11 gene under the control of the tetr...
The tetracycline repressor (TetR) from Escherichia coli was used to develop a system in which expression of herpesviral genes could be regulated. To develop the TetR-mediated gene repression system in HCMV, recombinant virues containing the US11 gene under the control of the tetracycline operator (tetO)-modified US11 promoter were isolated: DNA blot analysis attested to the fidelity of the recombination. Western blot experiment using polyclonal antisera of US11 gene product indicated that US11 gene expression of the recombinant virus was induced in the virus infected tTA expressing cell lines by the presence of tetracycline. The tTA-mediated gene activation system was examined in virus infected cells to determine its role in the control of gene expression. In the presence of tTA, the gene expression from the tetO-modified minimal promoter was efficiently activated in the uninfected cells, whereas essentially no activation was observed from the only minimal promoter without the seven direct repeats of 42 bp tetO sequences. On the other hand, in the infected cells, a substantial background of β -glucuronidase expression was detected in the absence of tTA, even though tTA stimulated the gene expression by about 7-fold. In tTA-mediated transcription activation system, homodimer consisted of tTA/tTA activates the tetO modified minimal promoter. To determine the effect of the heterodimer consisted of tTA/TetR on transcriptional activation from the minimal promoter, cotransfection assays were performed with U373 cells. In the cells expressing both tTA and TetR, the reporter g-ene expression from the minimal promoter was activated 4-12 fold higher than in the cells expressing only tTA, suggesting the higher efficacy of the tTA/TetR heterodimer on tTA-mediated transcription activation system. All these results suggest that tTA-mediated transcriptional repression system developed in this study has a potential applications to study the biological roles of HCMV genes. However, tTA-mediated transcriptional activation system may not be useful for studying the biological roles of HCMV due to the background stimulation by HCMV. The usage of the heterodimer consisted of tTA/TetR can improve the efficacy of tTA-mediated transcriptional activation system.
The tetracycline repressor (TetR) from Escherichia coli was used to develop a system in which expression of herpesviral genes could be regulated. To develop the TetR-mediated gene repression system in HCMV, recombinant virues containing the US11 gene under the control of the tetracycline operator (tetO)-modified US11 promoter were isolated: DNA blot analysis attested to the fidelity of the recombination. Western blot experiment using polyclonal antisera of US11 gene product indicated that US11 gene expression of the recombinant virus was induced in the virus infected tTA expressing cell lines by the presence of tetracycline. The tTA-mediated gene activation system was examined in virus infected cells to determine its role in the control of gene expression. In the presence of tTA, the gene expression from the tetO-modified minimal promoter was efficiently activated in the uninfected cells, whereas essentially no activation was observed from the only minimal promoter without the seven direct repeats of 42 bp tetO sequences. On the other hand, in the infected cells, a substantial background of β -glucuronidase expression was detected in the absence of tTA, even though tTA stimulated the gene expression by about 7-fold. In tTA-mediated transcription activation system, homodimer consisted of tTA/tTA activates the tetO modified minimal promoter. To determine the effect of the heterodimer consisted of tTA/TetR on transcriptional activation from the minimal promoter, cotransfection assays were performed with U373 cells. In the cells expressing both tTA and TetR, the reporter g-ene expression from the minimal promoter was activated 4-12 fold higher than in the cells expressing only tTA, suggesting the higher efficacy of the tTA/TetR heterodimer on tTA-mediated transcription activation system. All these results suggest that tTA-mediated transcriptional repression system developed in this study has a potential applications to study the biological roles of HCMV genes. However, tTA-mediated transcriptional activation system may not be useful for studying the biological roles of HCMV due to the background stimulation by HCMV. The usage of the heterodimer consisted of tTA/TetR can improve the efficacy of tTA-mediated transcriptional activation system.
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