臺灣博碩士論文加值系統

TACC3為Transforming Acidic Coiled-coil protein的簡稱。它與它的family proteins共同含有一段預報會形成盤繞狀的200酸性氨基酸。而之前也有文獻指出它在較快速分裂的細胞與一些癌細胞的表現比較高。目前其生理作用大多被報導具有穩定microtubules的功能，但是漸漸的越來越多文獻指出TACC3同時扮演調控基因transcription factor的角色。

在這裡我們主要是探討TACC3在不同細胞週期中，表現量的變化與被調控的相關機制。我們分別利用thymidine將細胞停留在G1/S phase，nocodazole把細胞停留在G2/M phase。在實驗中我們發現TACC3蛋白質的表現量會隨著時間形成一個鐘形曲線，TACC3的表現量在細胞週期中的M phase達到最高值。我們同時也觀察到TACC3在transcription level, mRNA的表現量亦在M phase達到最高值;另一方面在transcription level mRNA的穩定度是否會隨著細胞週期而改變，因此我們進一步使用actinomycin D抑制新合成RNA的產生，以利觀察mRNA的穩定度。實驗結果顯示RNA stability在TACC3細胞循環的表現不扮演重要的角色。經由實驗證實TACC3表現隨著細胞週期而改變，於是我们進一步探討細胞週期對於TACC3基因啟動區活性是否有所影響及主要的區段。藉由報告基因活性分析實驗，我們發現-586至+28基因啟動區序列具有調控TACC3在細胞週期中表現量的變化。值得注意的是，經由定序分析發現基因啟動區序列含有Sp1及CDE的結合位置，但仍有待進一步的釐清。

TACC3 was identified as a member of the transforming acidic coiled-coil (TACC) family characterized by their highly homologous carboxyl-terminal acidic coiled-coil domain. This protein has been found to be overexpressed in differentiating cells such as erythroid cells or in fast dividing cells such as cells in testis.

One of its most studied biologicalfunctions so far is the stabilization of centrosomal microtubules during M phase. However the mechanism of TACC3 expression during the cell cycle is yet not well understood. Here, we used Thymidine and Nocodazole to synchronize cells at G1/S and G2/M phase respectively, to study TACC3’s gene expression. Both TACCs RNA and protein expression showed a cyclical pattern over cell cycle. The importance of the fluctuations of TACC3 mRNA in the cell cycle raised the question of how it is regulated. Since the TACC3 mRNA stability between interphase and mitosis cells did not differ significantly, thus we proceeded to study the transcriptional regulation. Therefore, about 600 base pairs upstream of the 5’ untranslated region was cloned into pGL2-luciferase reporter vector. Our luciferase activity data showed that this region portrays a cyclical pattern, thus may contain cell cycle dependent regulatory elements. The deletion fragment, we identified two regions that play important role in the basal activity and activation of TACC3 during mitosis contained Sp1 and CDE binding sites. Further experiments will need to be done to confirm whether Sp1 bind to this region and which regulatory elements bind to the CDE binding sites.

Table of Contents

考試合格證明 -------------------------------------------------- I
Abstract ----------------------------------------------------------- II
Abstract in Chinese --------------------------------------------- III
Acknowledgement ---------------------------------------------- IV
Table of Contents ----------------------------------------------- V
Abbreviations ---------------------------------------------------- VII
List of Figures --------------------------------------------------- VIII


Chapter 1. Introduction ---------------------------------------- 1
I. TACC proteins ------------------------------------------------ 1
II. Cell cycle ----------------------------------------------------- 2
III. Cyclins and cyclin-dependent kinases ----------------- 3
IV. Cell cycle synchronization with Thymidine
and Nocodazole ------------------------------------------------- 3
V. RNA stability ------------------------------------------------ 4
VI. Sp1 of Sp Protein Family --------------------------------- 5
VII. CDE and CHR elements --------------------------------- 5
VIII.Aim ---------------------------------------------------------- 7


Chapter 2. Materials and Methods -------------------------- 13
I. Cell lines ------------------------------------------------------ 13
II. Antibodies --------------------------------------------------- 13
III. Materials ---------------------------------------------------- 13
IV. Reagent Preparation -------------------------------------- 18
V. Methods ------------------------------------------------------ 22
1. Cell Culture -------------------------------------------------- 23
2. Cell synchronization and 35S Methionine labeling ---23
3. Reverse Transcription Polymerase Chain Reaction -- 24
4. Western Blotting -------------------------------------------- 24
5. Construct ----------------------------------------------------- 24
6. Plasmid preparation ---------------------------------------- 25
7. Transfection with LipofectAMINE and Reporter
Gene Assay ---------------------------------- 26


Chapter 3. Results ---------------------------------------------- 27
I. TACC3 protein expression is increased during
G2/M phase ----------------------------------------------------- 27
II. Newly synthesized TACC3 protein labeled with
35S Methionine ------------------------------------------------ 28
III. TACC3 mRNA synthesis is cell cycle dependent --- 29
IV. Comparing TACC3 mRNA stability between
Nocodazole treated and control cells ----------------------- 30
V. Cloning of the 5’upstream region of the TACC3 gene - 31
VI. Study of the TACC3 promoter deletion fragments -- 32


Chapter 4. Discussion --------------------------------------- 34
References ---------------------------------------------------- 37
Figures --------------------------------------------------------- 43
Curriculum Vitae --------------------------------------------- 54

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