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系統識別號 U0026-0812200913360256
論文名稱(中文) 粒線體核醣體蛋白S36之研究
論文名稱(英文) Studies on mitochondrial ribosomal protein S36
校院名稱 成功大學
系所名稱(中) 基礎醫學研究所
系所名稱(英) Institute of Basic Medical Sciences
學年度 95
學期 1
出版年 96
研究生(中文) 陳永昌
研究生(英文) Yeong-Chang Chen
電子信箱 s5888122@mail.ncku.edu.tw
學號 s5888122
學位類別 博士
語文別 英文
論文頁數 64頁
口試委員 口試委員-林以行
口試委員-賴明德
指導教授-吳昭良
口試委員-戴明泓
口試委員-張文粲
口試委員-林清淵
中文關鍵字 前胸腺素  核醣體蛋白S36 
英文關鍵字 prothymosin  mitochondrial ribosomal protein S36 
學科別分類
中文摘要 細胞的生長、分化、發育都需要能量。粒腺體為細胞能量的來源。核醣體的生物活性與細胞循環、增生及腫瘤生成有關。有研究指出抑癌基因或原致癌基因會參與調控核醣體成熟與生物活性。抑癌基因p53受到核醣體蛋白或其他蛋白的修飾而引起細胞循環停滯或死亡。粒腺體的rRNA由粒腺體DNA轉錄而來,粒腺體核醣體蛋白的產生則來自細胞核基因。粒腺體核醣體蛋白的功能尚未被釐清而其是受到是轉錄因子嚴格的控制並與粒腺體的功能有關。我們選殖出一基因為老鼠粒腺體核醣體蛋白S36。我們將老鼠粒腺體核醣體蛋白S36送到NIH3T3中,發現老鼠粒腺體核醣體蛋白S36分佈於粒腺體中。老鼠粒腺體核醣體蛋白S36過量表現會造成抑癌基因p53的磷酸化並讓其下游基因p21表現進而引起細胞循環的停滯且影響粒腺體的功能。前胸腺素(ProT)過量表現會促進細胞的增生。前胸腺素是一個轉錄因子,其也可與其他轉錄因子如CBP結合調節基因的表現。我們的研究發現前胸腺素會造成粒腺體核醣體蛋白S36 mRNA的表現量增加。報告基因的研究也顯示出老鼠粒腺體核醣體蛋白S36啟動區段-480到-190 bp可能是前胸腺素的反應區域。在老鼠粒腺體核醣體蛋白S36啟動區段我們發現了可能是前胸腺素結合的片段,而其附近也含有其他其他轉錄因子如CREB的結合片段。我們推測前胸腺素可以透過與其他轉錄因子的作用來調控老鼠粒腺體核醣體蛋白S36的表現。
英文摘要 Most of the energy required for cell growth, differentiation, and development are met by mitochondrial ATP. Ribosomal biogenesis is correlated with cell cycle, cell proliferation, cell growth and tumorigenesis. Some oncogenes and tumor suppressors are involved in regulating the formation of mature ribosome and the ribosomal biogenesis. p53 induced cell cycle arrest when stabilized by ribosomal proteins and MDM2/HDM2 interaction. p53-dependent cycle arrest is primarily mediated by the CDK inhibitor p21. The mitochondrial rRNA are encoded by mtDNA, whereas the ribosomal proteins are encoded in the nuclear genes, they are synthesized on cytoplasmic ribosomes, and imported into the mitochondria. All of the proteins in mammalian mitochondrial ribosomes are products of nuclear genes. The majority of the mammalian MRPs have never been characterised in the laboratory. It is tightly regulated by series of transcription factors for maintain mitochondrial biogenesis and function. In this report, we cloned a gene mitochondrial ribosomal protein S36 (mMRPS36). Overexpression mMRPS36 in cells suppresses cell proliferation and induced cell cycle arrest. mMRPS36 overexpression induced p21 expression through the transcriptional level by p53 mechanism. We discovered that mMRPS36 protein is localized in the mitochondria and affects the mitochondrial function. These results suggest that mMRPS36 plays an important role in mitochondrial ribosomal biogenesis may cause nucleolar stress, leading to cell cycle arrest in a p21-dependent manner. Prothymosin α (ProTα) overexpression promotes cell proliferation and cell cycle. The function of ProTα in transcription activation interacted with co-activator of transcription CBP is a role for this acidic polypeptide in gene expression. In the present study, we found that ProTα increased the expression of mMRPS36 mRNA. Reporter assays revealed that the ProTα response region was in the range of -480 to -190 bp. ProTα is a nuclear protein and suggest that binds DNA in a sequence-specific manner. Potential ProTα binding sites were detected in 5’-flanking region of MRPS36 gene and closed to CREB transcription factor binding site. Taken together, these results suggest that C/EBP and CREB sites in the essential promoter region are critical for ProT response, and CREB showed a functional cooperation with coactivator p300/ CBP in driving the transcriptional regulation of ProT induce mMRPS36 gene expression.
論文目次 1.Qualified Certificate I
2.Acknowledgements II
3.Chinese Abstract III
4.English Abstract IV
5.Content Table VI
6.Index of Figures VIII
7.Abbreviations IX
8.Introduction 1
9.Specific aims 7
10.Materials and Methods 8
A. Material 8
A.1. Plasmids 8
A.2. Oligonucleotides 9
B. Method 9
B.1 Cell and Cell Culture 9
B.2 Representational Difference Analysis (RDA) 10
B.3 RT-PCR 10
B.4 Generation of mMRPS36-overexpression NIH3T3 Cells 11
B.5 Confocal Microscopy 11
B.6 Cell proliferation and Viability 12
B.7 Cloning of 5'-flanking region of mMRPS36 13
B.8 Prothymosin α DNA binding : random oligonucleotide selection 14
B.9 Template-repeated PCR (TR-PCR) 14
B.10 Luciferase Activity Assay 14
B.11 Detection of Transgene Expression in the Transfectants 15
B.12 Measurement of Mitochondrial Membrane Potential 18
B.13 Measurement of Reactive Oxygen Species (ROS) 18
11.Results 19
Specific RDA products 19
1. Function of Mitochondrial ribosomal protein S36 19
2. Regulation of mMRPS36 gene expression 24
12.Discussion 30
13.Conclusion 38
14.References 39
15.Appendix of Figures 48
16.Published articles
17.Curriculum Vitae
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