臺灣博碩士論文加值系統

Notch訊息傳導之路徑在動物體發育過程對於細胞命運之決定扮演著極為重要的調控角色。Notch訊息傳導途徑包含了Notch受體蛋白、Notch受質蛋白、細胞內作用分子與Notch修飾分子。Notch家族在演化上一直都被保留且維持相同功能，皆為穿越細胞膜一次之受體而且可直接調節基因的轉錄作用。
Notch受體蛋白包含有細胞外片段以及穿透膜-細胞內片段。細胞外片段包含有很多重複性的epidermal growth factor (EGF)-like區域，EGF重複區之後緊接著三個重複的Lin-12/Notch區域；細胞內片段在靠近細胞膜部位緊鄰著一段RAM23區域，其後接著包含了六個重複性的ankyrin區域、nuclear localization signal (NLS)片段、TAD (transcriptional activation domain)以及glutamine, serine and threonine rich (PEST) 區域。當Notch受體蛋白與標的物結合之後，會促使其被蛋白酵素作用，造成Notch受體之活化形 (NICD) 即細胞內片段之釋放，而此Notch受體之活化形會被轉送至細胞核內，接著與位在核內之DNA結合蛋白CBF1進行交互作用，進而使一些與細胞分化有關之基因進行轉錄作用。Notch受體活化形與CBF1蛋白形成之複合物會和許多調節因子結合，例如：活化因子或是抑制因子。但至現今，這些因子的作用機制仍尚未明瞭且仍需要更深入之研究與探討。
本實驗室為了尋找參與CBF1-dependent Notch訊息傳遞途徑之新調節因子並研究其作用機制，因此需建立一個良好之實驗分析系統。我們參考在其他系統中之Notch1受體活化形而設計出四種短縮形之人類Notch1分子。下列為四種短縮形Notch1分子之胺基酸位置：(1) ANK 重複區，nt 5461-6615 (amino acid 1820-2205)；(2) RAM+ANK，nt 5290-6615 (amino acid 1763-2205)；(3) ANK+TAD，nt 5461-7332 (amino acid 1820-2444)；及(4) RAM+ANK+TAD，nt 5290-7332 (amino acid 1763-2444)。
此四種短縮形Notch1之細胞內區域之N端皆標有myc-tag；ANK1-6重複區與RAM至ANK1-6重複區之C端則另外標示了His-tag。將此4種短縮形之Notch1 cDNA轉殖至pcDNA3表現質體上而獲得了下列四種質體：pcDNA3/a, pcDNA3/ra, pcDNA3/ac, and pcDNA3/rac，接著利用calcium phosphate沉澱之方法將其轉染至COS7和HEK293細胞中。為了分析內生性CBF1活性之變化，除了表現質體之外，我們將含有CBF-1作用子結合序列之p(FPⅢ)6 CAT報導質體，以及組間對照質體pcDNA3.1/myc-his/LacZ.一起協同轉染至細胞中。
在本論文中，我們利用anti-myc, anti-His初級抗體及anti-Notch1 C-terminal初級抗體以西方點墨法確認轉染至COS7和HEK293細胞中之4種短縮形Notch1分子皆有表現且其分子量大小正確 (ANK1-6重複區分子量約為60 kDa；RAM至ANK重複區約為70 kDa；ANK重複區至C端位置TAD區域之分子量約為95 kDa；RAM區域至C端TAD區域分子量約為100 kDa)。在轉染48小時後萃取轉染細胞之細胞液進行chloramphenicol acetyl transferase (CAT) 活性之檢測。將各組Notch1短縮形之CAT分析所得之結果先經由β-galactosidase的活性進行校正後，再與不含Notch1短縮形之pcDNA3表現質體控制組的CAT活性值做比較。實驗結果顯示，人類Notch1之ANK重複區的DNA序列並不會影響CBF1作用子之活性，但含有RAM區域或C端TAD區域時則會對CBF1之轉錄活性產生影響。與控制組相比，RAM至ANK重複區的DNA片段可以增加CBF1作用子之轉錄活性約3倍；ANK重複區至C端TAD區域組則可以增加CBF1作用子之轉錄活性約5倍；而RAM至C端TAD區域組則可顯著地增加CBF1作用子的活性約12倍。此結果產生之原因是因為RAM區域主要功能為與CBF1作用子結合的部位；ANK區域與CBF1作用子有少部分之結合；C端的TAD在Notch受體蛋白中扮演著活化作用的角色。

The Notch signaling pathway is a ubiquitous pathway that controls a variety of processes during development. Notch signaling pathway involves Notch receptors, Notch ligands, intracellular effectors and Notch modulators. The Notch family comprises a group of highly conserved proteins that function both as cell surface receptors and direct regulators of gene transcription.
The extracellular domain of Notch receptor is composed of epidermal growth factor-like repeats as well as three repeats designated LNR repeats. Further, the intracellular domain of Notch contains six copies of an ankyrin-like motif, NLS (nuclear localization signal) sequence, TAD (transcriptional activation domain), and PEST (glutamine, serine and threonine rich) domain. The full-length Notch receptor undergoes a proteolytic cleavage event upon ligand binding which potentiates the relocation of its intracellular domain (NICD) to the nucleus and interacts with the DNA binding protein CBF1 to activate transcription of genes that regulate cell differentiation. There are regulatory proteins interact with this NICD-CBF1 complex to function as activators (e.g mastermind) or repressors. The molecular mechanisms involved have been a focus of intense research and remain controversial.
We established a co-transfection assay system in order to search and study the mechanisms of novel factors that affect the CBF1-dependent Notch signaling. Four truncated human Notch1 molecules comparable to activated Notch molecules known to be functional in other systems were used in this study. The nucleotide position of these four truncated molecules in Notch1 receptor are following. (1) ANK repeats, nt 5461-6615 (amino acid 1820-2205); (2) RAM domain and ANK repeats, nt 5290-6615 (amino acid 1763-2205); (3) ANK repeats and the C-terminal TAD, nt 5461-7332 (amino acid 1820-2444); and (4) RAM+ANK+ TAD, nt 5290-7332 (amino acid 1763-2444).
The N-terminal of all truncated human Notch1 constructs were tagged with myc-tag, the C-terminal of ANK repaets and RAM+ANK were also tagged with His-tag. The cDNA fragments were then subcloned into the mammalian expression plasmid pcDNA3 to obtain four plasmids: pcDNA3/a, pcDNA3/ra, pcDNA3/ac, and pcDNA3/rac. The calcium phosphate precipitation method was used to transfcet plasmids into COS-7 and HEK293 cells. To assay for the activation of endogenous CBF1, besides plasmids of Notch1 construct, cells were cotransfected with p(FPⅢ)6 CAT reporter plasmid and the internal control plasmid pcDNA3.1/myc-his/LacZ.
The expression of truncated Notch proteins with correct molecular weights in COS7 and HEK293 cells were confirmed by Western blot analysis using anti-myc, anti-His antibodies, and anti-Notch1 C-terminal antibodies (ANK repeats≒60 kDa；RAM domain+ANK repeats≒70 kDa；ANK+C-terminal TAD≒95 kDa；RAM+ANK +TAD≒100 kDa). All transfceted cells were harvested 48 hours after DNA transfection and chloramphenicol acetyl transferase (CAT) activities were measured. Relative activation of the reporter construct was calculated as fold activation compared to empty vector (pcDNA3) transfection and normalized for the activity of b-galactosidase. Although the CBF1 protein with the human Notch1 ANK repeats failed to activate transcription, the activity was recovered by addition of either RAM domain or TAD. Relative to the control, CBF1 activated expression was increased ~3-fold by the RAM+ANK fragment, ~5-fold by the ANK +TAD, and ~12-fold by the RAM+ANK +TAD fragment. The result of CAT assay reflects the fact that the RAM domain is the primary binding domain to CBF1, ANK repaets interact weakly with CBF1, and TAD is important for transactivation activity.

中文摘要……………………………………………………………………………..i
英文摘要……………………………………………………………………………..iii
目次…………………………………………………………………………………...v
壹、緒論
一、Notch受體蛋白的結構………………………………………………….1
二、DSL 標的物之結構……………………………………………………..3
三、Notch受體蛋白的活化………………………………………………….3
四、CSL作用子……………………………………………………………...5
五、Notch之訊息傳遞路徑………………………………………………….6
(一)CSL-dependent pathway…………………………………………….7
(二)CSL-independent pathway…………………………………………..7
六、Notch訊息之調節……………………………………………………….8
(一)Mastermind………………………………………………………….9
(二)Fringe………………………………………………………………..9
(三)Dishevelled………………………………………………………...10
(四)Numb……………………………………………………………....10
七、關於Notch受體細胞內區域之研究…………………………………...10
貳、實驗目的……………………………………………………………………...14
參、實驗材料與方法……………………………………………………………..15
一、重組表現質體DNA的製備…………………………………………...16
(一)DNA片段的透析膜電泳純化……………………………………16
(二)XL10-Gold勝任性細菌(Competent cell)之製備…………………17
(三)接合反應 (ligation)……………………………………………..….18
(四)大腸桿菌之轉殖作用 (Transformation for E. coli)……………..…19
(五)鑑定轉殖基因………………………………………………………20
(六)基因的定序…………………………………………………………20
二、細胞的培養………………………………………………………………21
三、西方點墨法……………………………………………………………....21
四、轉染用之重組表現質體DNA的製備…………………………………..22
五、細胞培養之磷酸鈣短暫轉染
(Calcium phosphate transient transfection for cell culture)………………23
六、細胞液之萃取……………………………………………………………24
七、β-galactosidase活性之檢測……………………………………………25
八、鑑定表現質體之表現……………………………………………………25
九、Chloramphenicol acetyl transferase (CAT) 活性的檢測………………..25
肆、實驗結果……………………………………………………………………...27
一、檢測Notch受體活化形之存在…………………………………….…...27
二、表現質體之構築……………………………………………………..….27
(一)製備含ANK1-6重複區之表現質體………………………………27
(二)製備含有RAM至ANK1-6重複區之表現質體………………….27
(三)製備含有ANK 1-6重複區至C端位置TAD區域之表現質體….28
(四)製備含有RAM區域至C端TAD區域之表現質體……………...28
三、鑑定表現質體之表現……………………………………………………29
(一)表現質體在COS-7細胞中之表現………………………………..29
(二)表現質體在HEK293細胞中之表現………………………………30
四、短縮形Notch1對CBF1作用子活性之影響…………………………...30
伍、結果討論………………………………………………………………………32
一、關於實驗之設計……………………………………………………….32
二、Notch1短縮形之表現………………………………………………….33
三、RAM區域至C端TAD區域對CBF1轉錄活性有最顯著的影響…..35
陸、參考文獻……………………………………………………………………..38
柒、附錄…………………………………………………………………………..47

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