臺灣博碩士論文加值系統

p53是一個腫瘤抑制基因， 其主要功能在維持細胞基因體的完整性。 本實驗室曾利用mRNA差異表現法找到兩個可以受p53活化的基因， mDDA1及mDDA3。 同時在EST database中找到了mDDA3在人類的相似物， hDDA3。 mDDA3與hDDA3的胺基酸序列中分別含有六個及三個的PXXP motif， 而且都含有一個可能會形成coiled-coil region的序列。 由於coiled-coil region與PXXP motif都可以媒介與其他蛋白質的相互結合， 所以我們以yeast two-hybrid的方法嘗試找尋與DDA3結合的蛋白， 希望能藉由對DDA3結合蛋白的研究而對DDA3的功能有進一步的瞭解。
以yeast two-hybrid的方法， 我們找到了許多可能與DDA3相結合的蛋白， 其中之一為小鼠53BP2 (m53BP2)。 進一步的研究發現mDDA3能夠與m53BP2， 人類53BP2 (h53BP2) 以及其相似物KIAA0771的蛋白質之C端相結合， 但無法與全長之h53BP2以及其相似物KIAA0771結合。 hDDA3則能夠與這些蛋白的C端以及全長相結合。 而且將N端包含兩個PXXP的序列刪除後的hDDA3仍然能夠與53BP2相結合， 表示DDA3與53BP2的結合不需要其N端的兩個PXXP motif。
對hDDA3與h53BP2在細胞內分佈的研究發現hDDA3表現在細胞質以及細胞核中， 而且在核中呈絲狀結構。 53BP2在細胞內則散佈於細胞質中。 若將hDDA3與53BP2同時表現在細胞內， 則可以看到53BP2有部份分佈於細胞核附近。
53BP2可以增強p53的轉錄活化功能。 我們利用luciferase assay發現53BP2可以將p53的轉錄活化功能增加至2.5倍， 但hDDA3對53BP2此一作用似乎沒有影響。 所以hDDA3與53BP2相結合所產生的影響尚需繼續的研究。

p53 is a tumor suppressor gene that helps to maintain the integrity of the genome. Through the method of mRNA differential display, we have previously found two p53 inducible mouse genes, mDDA1 and mDDA3. The human homologue of mDDA3 has been found and designated hDDA3. The amino acid sequences of hDDA3 and mDDA3 contain 3 and 6 PXXP motifs respectively, and both include a putative coiled-coil region. Since PXXP motif and coiled-coil region may mediate protein-protein interactions, we utilized a yeast two-hybrid assay to screen for mDDA3-interacting proteins from mouse brain cDNA library.
Out of several mDDA3-interacting proteins, 53BP2 is of particular interest. Further yeast two-hybrid analyses have revealed that mDDA3 interacts with the C-terminus of mouse and human 53BP2 and its human homologue KIAA0771 but failed to interact with the full-length 53BP2 proteins and KIAA0771. hDDA3 can interact with both the C-terminus and full length of 53BP2 proteins and homologues. We also found that deletion of the N-terminal region containing 2 PXXP motifs of hDDA3 does not interfere with its 53BP2 binding ability.
Immunofluorescence studies revealed that hDDA3 is localized in both the cytoplasm and the nucleus. 53BP2 is present mainly in the cytoplasm but when co-expressed with hDDA3, a fraction of 53BP2 protein seems to relocate to the perinuclear region.
53BP2 has been found to stimulate the transactivation activity of p53. But utilizing a luciferase assay, hDDA3 didn't seem to affect the stimulation of p53 by 53BP2. The functional significance of 53BP2-hDDA3 interaction thus remains to be elucidated.
中文摘要……………………………………1
英文摘要……………………………………2
壹. 緒論……………………………………3
貳. 實驗材料與方法………………………11
參. 實驗結果………………………………25
肆. 討論……………………………………33
伍. 圖表……………………………………40
陸. 參考文獻………………………………57

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