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研究生:張長齡
研究生(外文):Chang-Lin Chang
論文名稱:人類Chk1基因表現與調控機制之探討
論文名稱(外文):Characterization of Human Chk1 Gene Expression and Regulation
指導教授:徐明達徐明達引用關係
指導教授(外文):Ming-Ta Hsu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:100
中文關鍵詞:細胞週期
外文關鍵詞:Chk1checkpointcell cycle
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細胞生長的過程中需要保持基因體的完整性,避免細胞發生變異、死亡,以及癌化。然而,細胞經常受到外在環境各種刺激或傷害,如紫外光、放射線、化學藥劑等,其本身也會因代謝過程中產生過氧化物及複製錯誤等造成對Genomic DNA的傷害。Checkpoint 的功用為偵測細胞內DNA damage的發生,並藉由阻止細胞週期的進行,提供細胞足夠的時間修復錯誤,Chk1即為其中的一種重要的基因。Chk1主要參與G2/M checkpoint,也抑制G1/S及intra S arrest的進行。我們以分化前後U937細胞為模型,探討Chk1在人類細胞中轉錄產物的多樣性及調控機制。我們發現在U937及其他細胞中Chk1具有兩種不同的transcript,其分別含有兩種不同的 leader Exon,Exon N1及Exon 1,造成含有不同的5’ UTR。兩種transcript除了表現量不同外,含有Exon N1的 transcript還具有一種沒有Exon 3的異構RNA的存在。我們在人類組織、胚胎組織、血球細胞中看到兩種transcript有不同表現量,暗示兩者以不同轉錄機制調控其表現;若以PMA促使U937細胞分化,亦發現兩種transcript受到藥物處理前後的表現變化並不一致。我們分別對兩者進行研究,發現含有Exon N1的Chk1 transcript可能以直接或間接的方式受p53的抑制;而含有Exon 1的Chk1 transcript的調控機制較不清楚,僅知可能是由PKC下游的訊息傳遞所調控,且可能和MAP kinase cascade有所關聯。另外,我們以polysome gradient的技術發現U937細胞分化後E1 transcript的轉譯效率上升,暗示著細胞可能在不同時期選擇不同transcript以調控Chk1的蛋白表現。最後,我們對Chk1進行3’ UTR的分析與實驗後,發現了五種不同形式的3’ UTR,也說明了人類Chk1在轉錄時期的多樣性。綜合以上所述,本實驗提出了人類Chk1在轉錄產物的多樣性,以及可能的轉錄調控機制。雖然還有許多尚待釐清的部分,也許藉由本實驗的結果,有助於對人類Chk1在轉錄及轉譯時期的調控有所了解。
Maintenance of genomic integrity is essential for cell proliferation and development. Genomic instability may result in mutation, apoptosis, or tumorgenesis. However, the genome of eukaryotic cells is often assaulted by the environmental factor such as UV, IR, and chemical reagent as well as damaged by the byproducts of normal intracellular metabolism (e.g., reactive oxygen intermediates). Checkpoint can detect DNA damage and arrest cell cycle progression to provide time for DNA repair. Chk1 is one of the major checkpoint gene involved in G2/M checkpoint, as well as involved in G1/S and Intra S arrest. We use U937 human monoblastic leukemia cell as a model to study human Chk1 RNA isoforms and transcription regulation. We found that Chk1 has two different leader exons, Exon N1 and Exon 1.The RNA contains these leader exons These two transcripts are different in expression level and the transcript containing Exon N1 has an isoform that skips exon 3. In addition, these two transcripts have different expression levels in human adult tissues, human embryonic tissues, and human leukocyte. Furthermore, we observed that the expression of the two types of transcript oppositely regulated by PMA treatment. In our study, we found that the transcript contains N1 leader exon may be regulated indirectly by p53-dependent mechanism. However, the regulatory mechanism of E1 transcript remains unclear. Our results indicate that the RNA species may be regulated by PKC downstream signals related to the MAP kinase cascade. We observed E1 contains RNA increase in the polysome after induction of U937 differentiation. This result suggests that differ of Chk1 isoforms may be used for synthesizing Chk1 protein during U937 differentiation. Finally, we analyzed 3’ UTR region of Chk1, and found five different kinds of 3’ UTR. In summary, this work has shown that human Chk1 gene contain heterogeneous population of RNA isoforms. Further study is needed to address the biological functions and regulation of synthesis of these Chk1 RNA isoforms.
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