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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 吳益群 | |
dc.contributor.author | Ya-Yin Tsai | en |
dc.contributor.author | 蔡雅因 | zh_TW |
dc.date.accessioned | 2021-07-10T21:34:42Z | - |
dc.date.available | 2021-07-10T21:34:42Z | - |
dc.date.copyright | 2016-11-02 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-19 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76663 | - |
dc.description.abstract | 在動物體的發育過程中,計畫性細胞凋亡是一個具有高度保留性的過程。在線蟲當中,當egl-1/BH-3-only gene的轉錄作用啟動,會促使大部分的體細胞進行細胞凋亡,然而在尾突細胞中,會透過另一條途徑活化計畫性細胞凋亡。目前已知尾突細胞會透過PAL-1,這個轉錄因子結合至ced-3的啟動子,促使ced-3轉錄,使尾突細胞進行計畫性細胞凋亡,而這個過程證明細胞凋亡的啟動可以是和egl-1無關。然而,在沒有egl-1的時候如何調控細胞死亡的時間,其機制不太清楚。根據我們實驗室先前的研究發現,BLMP-1,一個鋅手指轉錄抑制因子,會參與在尾突細胞凋亡當中。同時發現在尾突細胞死亡前BLMP-1才開始表現,這表示blmp-1的表現時間點是被嚴格控制的,而且在尾突細胞凋亡中扮演重要的角色。為了瞭解blmp-1在計畫性細胞凋亡過程當中所扮演的角色,我們尋找其下游的因子。藉由遺傳分析發現,在blmp-1突變中,尾突細胞的異常存活需要ced-9/bcl-2。此外,利用dGFP在ced-3(n717)突變中的表現,ced-3(n717)是讓細胞死亡有缺陷的突變,讓尾突細胞得以存活下來讓我們觀察,我們發現ced-9在尾突細胞死亡之前開一直表現。而在ced-3(n717); blmp-1(RNAi)突變中,ced-9持續表現。同時我們也發現兩個(Pax)轉錄因子,會在生殖細胞促進ced-9的表現來保護細胞免於細胞凋亡,但是不參與在blmp-1所調控的尾突細胞凋亡當中,這意味著,blmp-1有可能是直接調控ced-9的轉錄,而ced-9的啟動子也發現具有blmp-1的潛在結合序列。我們利用electrophoretic mobility shift assay (EMSA)來驗證是否BLMP-1鋅手指蛋白能夠結合到ced-9啟動子上,而我們發現BLMP-1鋅手指蛋白能夠結合到ced-9啟動子來調控尾突細胞的凋亡。DRE-1是一個在SCF(Skp1-Cullin-F-box) ubiquitin連接酶複合物中的F-box蛋白,DRE-1已經被研究會透過ced-9促進尾突細胞凋亡,而我們利用遺傳分析證明dre-1和blmp-1透過平行的路徑促進尾突細胞的死亡。綜合上述的結果,我們知道blmp-1在核心細胞凋亡路徑中的位置,而我們的研究結果提供了一個時間上經由ced-9調控計畫性細胞凋亡的機制。 | zh_TW |
dc.description.abstract | Programmed cell death (PCD) is a conserved cellular process during animal development. In Caenorhabditis elegans (C. elegans), transcriptional up-regulation of egl-1/BH-3-only gene promotes most somatic cell deaths, but tail-spike cell uses an alternative mechanism to initiate PCD where PAL-1, a transcription factor, binds to ced-3 promoter and re-initiates ced-3 transcription to promote tail-spike cell death, demonstrating that cell death onset could be egl-1-independent. Yet, little is understood about the temporal control mechanism of cell death in the absence of egl-1. Previously, our lab found that BLMP-1, a zinc finger transcriptional repressor, is required for the tail-spike death. The expression of BLMP-1 in tail-spike cell was observed just before its death, indicating that the timing of blmp-1 expression is tightly controlled and important for tail-spike cell death. To understand the role of blmp-1 in PCD, we looked for its downstream effectors. Our genetic analysis revealed that the abnormal survival of tail-spike cell in the blmp-1 mutant required ced-9/bcl-2. In addition, using dGFP transcriptional reporter in ced-3(n717) mutants as a death defective background which allow tail-spike cell to survive, we found that ced-9 is expressed in tail-spike cells just before its death. Moreover, ced-9 transcription persists in the tail spike cell in ced-3(n717); blmp-1(RNAi). These data indicated that ced-9 was repressed by blmp-1. While two paired box (Pax) transcription factors, pax-2 and egl-38, promote ced-9 transcription to protect cell from death in germline cells, we found that they were not involved in blmp-1-mediated tail-spike cell death, raising the possibility that blmp-1 may regulate ced-9 transcription directly. In support of this, the ced-9 promoter contains a potential BLMP-1 binding site. We employed electrophoretic mobility shift assay(EMSA) to test if BLMP-1 is able to bind to ced-9 promoter, we found that BLMP-1 Zn finger protein is able to bind to ced-9 promoter to mediate tail-spike cell death. DRE-1, the F-box protein of SCF (Skp1-Cullin-F-box protein) ubiquitin ligase complex, is previously identified to promote the tail-spike death through ced-9. Our epistasis analysis suggested that dre-1 acts in parallel of blmp-1 to promote the tail-spike cell. Taken together, we have characterized the position of blmp-1 in the core program cell death pathway. Our results provide a temporal regulatory mechanism of PCD through ced-9. | en |
dc.description.provenance | Made available in DSpace on 2021-07-10T21:34:42Z (GMT). No. of bitstreams: 1 ntu-105-R03b43016-1.pdf: 1783706 bytes, checksum: b9f04b42a5d37b11d7ed44c03d2b8dd8 (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 致謝................................................................................................................................... i
摘要.................................................................................................................................. ii Abstract .......................................................................................................................... iv Table of Contents............................................................................................................ 1 Introduction .................................................................................................................... 4 Materials and Methods ................................................................................................ 10 Strains..................................................................................................................... 10 Transgenic worms................................................................................................... 10 DNA constructs ...................................................................................................... 12 RAN interference(RNAi) ....................................................................................... 13 Quantify the GFP reporter signal............................................................................ 14 Purification of recombinant proteins...................................................................... 14 Probes..................................................................................................................... 14 EMSA (Electrophoretic mobility shift assay) ........................................................ 15 Results............................................................................................................................ 16 blmp-1 functions to promote the tail-spike cell death through ced-9..................... 16 ced-9 was expressed during early embryonic development and lost its expression in the tail spike cell before its death ........................................................................... 18 ced-9 is transcriptionally expressed in the tail spike cell before its death in ced- 3(n717); blmp-1(RNAi) .......................................................................................... 19 blmp-1 regulates neither pax-2 nor egl-38 in tail-spike cell death ......................... 20 Sequence analysis showed potential BLMP-1 binding sites in ced-9 promoter (2Kb)....................................................................................................................... 20 Deletion of a potential BLMP-1 binding site in ced-9 promoter affects the expression pattern of the ced-9 transcriptional reporter......................................... 21 BLMP-1 binds to specific regions of ced-9 promoter............................................ 22 dre-1 acts downstream or in parallel of blmp-1 to mediate tail-spike cell death.... 23 Discussion ...................................................................................................................... 25 blmp-1 regulates ced-9 expression to promote the tail-spike cell death................. 25 BLMP-1 may target at several different regions on ced-9 promoter...................... 26 dre-1 acts upstream of ced-9 and in parallel to blmp-1 .......................................... 27 References ..................................................................................................................... 29 Figure............................................................................................................................. 32 Figure 1. The programmed cell death pathway of C. elegans is evolutionarily conserved................................................................................................................ 32 Figure 2. Development of tail-spike cell at different stages................................... 33 ................................................................................................................................ 34 Figure 3. Current model for tail-spike cell death. .................................................. 34 Figure 4. Gene structure of blmp-1 and its potential binding sequence. ................ 35 Figure 5. The ectopic survival of tail-spike in blmp-1 mutants requires ced-9...... 36 Figure 6. ced-9 transcriptional reporter was expressed during early embryonic development and lost its expression in the tail spike cell before its death. ............ 38 Figure 7. The expression of ced-9 transcriptional reporter persists in the tail spike cell before its death in ced-3(n717); blmp-1(RNAi). .............................................. 39 Figure 8. blmp-1 regulates neither pax-2 nor egl-38 in tail-spike cell death.......... 40 Figure 9. Sequence analysis showed potential BLMP-1 binding sites in ced-9 promoter (2Kb)....................................................................................................... 42 Figure 10. Deletion of a potential BLMP-1 binding site in ced-9 promoter affects the expression pattern of the ced-9 transcriptional reporter. .................................. 44 Figure 11. MBP::BLMP-1 directly binds to specific regions of the ced-9 promoter in an EMSA assay. ................................................................................................. 45 Figure 12. Over-expression of DRE-1 can rescue the tail-spike cell death defect in blmp-1 mutants. ...................................................................................................... 46 | |
dc.language.iso | en | |
dc.title | blmp-1抑制ced-9轉錄作用促進線蟲尾突細胞凋亡 | zh_TW |
dc.title | blmp-1 suppresses ced-9 transcription to promote tail-spike cell death in C. elegans | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 潘惠錦,蔡欣祐 | |
dc.subject.keyword | 細胞凋亡,尾突細胞,blmp-1,細胞凋亡核心路徑, | zh_TW |
dc.subject.keyword | programmed cell death,tail-spike cell,blmp-1,core pathway, | en |
dc.relation.page | 46 | |
dc.identifier.doi | 10.6342/NTU201602822 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2016-08-20 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
顯示於系所單位: | 分子與細胞生物學研究所 |
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