周子賓臺灣大學：分子與細胞生物學研究所徐唯珊Hsu, Wei-ShanWei-ShanHsu2007-11-252018-07-062007-11-252018-07-062004http://ntur.lib.ntu.edu.tw//handle/246246/49894果蠅的CG11183最先被認為是人類SMIF基因的同源基因，而被命名為dSMIF。SMIF不但參與變形生長因子貝他(TGF-β)的訊息傳遞，也被證實是一個去頭蓋酵素，因此SMIF基因也被命名為hDcp1a。由於缺乏直接的遺傳證據證明CG11183為果蠅TGF-β訊息傳遞的成員，但是CG11183經證實後具有去頭蓋酵素活性，因此被命名為果蠅的去頭蓋酵素，dDcp1。 為了闡明dDcp1與TGF-β訊息傳遞之間的關聯性，我們以三個方向進行探討:一，以酵母菌融合實驗檢驗dDcp1與TGF-β訊息傳遞成員間的作用力關係。二，以P跳躍元素進行不準確的跳出跳動，取得無功能的dDcp1對偶基因。三，分析dDcp1無功能對偶基因所產生的性狀，期藉此得到直接的證據證實dDcp1為TGF-β訊息傳遞之成員。 經酵母菌融合實驗證實，dDcp1不但與果蠅的Co-Smad，Medea，具有相互作用的能力，且與果蠅的兩個R-Smad，Mad以及dSmad2，亦能相互作用；此外，dDcp1與Co-Smad以及R-Smad可以形成三複體。dDcp1也具有活化轉錄的能力，本身也能與自己形成複體。這些結果指出dDcp1很可能是一個TGF-β訊息傳遞中的轉錄共同調控蛋白。 經兩次P跳躍元素不準確的跳出篩選，dDcp1442pop被證實是一個沒有蛋白質表現的無功能對偶基因。dDcp1442pop使成蟲盤組織細胞以及幼蟲腦的視葉無法發育，並且幼蟲腦的EcR-B1蛋白質表現受到影響。這些現象顯示dDcp1很有可能是一個具有組織特異性之TGF-β訊息傳遞的轉錄共同調控蛋白。CG11183 is the Drosophila homology of SMIF and was named as dSMIF first. SMIF is identified as a transcriptional co-activator in transforming growth factor-β (TGF-β) signaling pathway and it also has decapping activity. Therefore, SMIF is also named as hDcp1a. However, there is no genetic evidence to link CG11183 with Drosophila TGF-β signaling. Moreover, CG11183 was later found to have the intrinsic decapping activity. Therefore, we named CG11183 as Drosophila decapping protein 1(dDcp1). In order to clarify the relationship between dDcp1 and TGF-β signaling pathway, we approached this aim in three aspects. First, to investigate the possible interaction between dDcp1 and the members in Drosophila TGF-β signaling pathway by yeast two/three-hybrid assay. Second, to create a dDcp1 null allele by imprecise excision screen. Third, to analyze the dDcp1 null phenotype and try to get the direct evidences to connect dDcp1 with TGF-β signaling. From the one/two/three-hybrid assay, we proved that dDcp1 can interact not only with the Drosophila Co-Smad, Medea, but also with the two Drosophila R-Smads, Mad and dSmad2. It was also certified that dDcp1, R-Smad and Co-Smad form a trimer. Further, dDcp1 is considered to have the ability to form oligomer and has the transactivation activity at the C-terminal. These results imply that dDcp1 is a putative transcriptional co-activator in TGF-β signaling. After two imprecise excision screens, dDcp1442pop was verified as a null allele. The imaginal discs and potic lobes can not development in dDcp1442pop larvae. Moreover, dDcp1442pop larvae displayed lower EcR-B1 expression in the central brain. These indicate that dDcp1 is a putative tissue-specific transcriptional co-activator in TGF-β signaling.中文摘要………………………………………………………………………………1 Abstract……………………………………………………………………………….2 Table of Content……………………………………………………………………...3 List of Tables………………………………………………………………………….6 List of Figures………………………………………………………………………...7 Introduction…………………………………………………………………………..8 Overview of Drosophila oogenesis………………………………………………8 Anterior-posterior axis determination of oocyte…………………………………9 Dorsal-ventral axis determination of oocyte……………………………………11 Overview of TGF-β signaling transduction…………………………………….12 TGF-β signaling in Drosophila…………………………………………………15 Roles of TGF-β signaling during Drosophila development…………………….16 dDcp1 is a putative multiple function protein…………………………………..18 dDcp1 is a novel posterior group gene………………………………………….18 The possible connection between dDcp1 and TGF-β signaling…………….......19 The themes……………………………………………………………………...20 Materials and Methods……………………………………………………………..22 Fly stocks and maintenance……………………………………………………..22 Imprecision excision screens……………………………………………………22 For b53 mutant……………………………………………………………….22 For P{GSV6}11684 allele……………………………………………………...23 Single fly PCR…………………………………………………………………..24 Germ-line clone production……………………………………………………..24 Cuticle preparation……………………………………………………………...24 Mis-expression experiment……………………………………………………..24 Immunohistochemistry………………………………………………………….25 For ovary……………………………………………………………………..25 For brain……………………………………………………………………...25 X-gal staining………………………………………………………………….. 26 Western analysis………………………………………………………………...26 Plasmid construction……………………………………………………………27 Yeast One-Hybrid……………………………………………………………….28 Yeast Two-Hybrid……………………………………………………………….28 Yeast Three-Hybrid …………………………………………………………….29 ONPG assay…………………………………………………………………….29 Result………………………………………………………………………………...31 dDcp1 interacts with R-Smads in yeast system……………………...................31 dDcp1, R-Smad and Co-Smad form a complex………………………………...36 dDcp1 mediates transcriptional activity in one-hybrid test……………………..38 dDcp1 form oligomer through the C terminal 74 amino acids………………….40 Isolation of dDcp1 null allele…………………………………………………...42 Imprecise excision of b53…………………………………………………….42 Imprecise excision of P{GSV6}11684 allele…………………………………..46 dDcp1442pop is a dDcp1 null allele………………………………………………48 Lethal phase of dDcp1 mutant organism………………………………………..50 dDcp1 is required for both imaginal discs and optic lobes development……….51 dDcp1442pop fails to upregulate the EcR-B1 expression in larval brain………....52 dDcp1442pop involves in the determination and posterior positioning of the oocyte ..............................................................................................................................53 Discussion....................................................................................................................55 Imprecise excision frequency...............................................................................55 dDcp1 is a putative co-activator in TGF-β signaling...........................................56 dDcp1 regulates the development of imaginal discs............................................58 dDcp1 controls the development of central nerve system....................................60 dDcp1 is a putative tissue-specific co-activator in TGF-β signaling...................61 dDcp1 involves in the determination and posterior positioning of the oocyte.....61 Acknowledgments.......................................................................................................64 Reference.....................................................................................................................651121866 bytesapplication/pdfen-US果蠅, TGF-βDrosophila, TGF-βotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/49894/1/ntu-93-R91225006-1.pdf