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標題: | DPY-24蛋白質層次在Distal Tip Cells遷移中的調控機制 Regulation of DPY-24 Level in Distal Tip Cells During Gonadogenesis of C. elegans |
作者: | Guan-Wei Ho 何冠緯 |
指導教授: | 吳益群(Yi-Chun Wu) |
關鍵字: | 線蟲,生殖腺,發育,基因調控, distal tip cell,gonadogenesis,development,gene regulation,protein stability, |
出版年 : | 2007 |
學位: | 碩士 |
摘要: | 在線蟲的hermaphrodites發育過程中,兩個distal tip cells (DTCs)會進行細胞遷移並使生殖腺發育為前後各一U字型的形狀,這個U字型的細胞遷移路徑分為三個階段:階段一,兩個DTC沿著腹側向線蟲身體兩側遷移至咽喉及肛門旁;階段二,DTC轉90度進行背向轉彎;階段三,DTC再轉90度並沿著背側遷移回到身體中央並停止。dpy-24基因表現出的蛋白質DPY-24具有一個PR domain及一個zinc finger,而這個protein藉由轉錄調控unc-5的表現來控制DTC進行dorsal turn的時間。dpy-24突變株會出現DTC提早進行dorsal turn的性狀。利用anti-DPY-24抗體進行免疫染色的結果顯示DPY-24蛋白質可以在DTC進行dorsal turn之前被偵測到,但是在轉彎之後就再也無法偵測到DPY-24信號。為了研究DPY-24 protein量在轉彎之後下降的原因,我們製造了許多轉錄與轉譯的GFP接合表現質體,發現由dpy-24 promoter驅動表現的GFP (Pdpy-24::gfp::unc-54 3’UTR)及與GFP接合的dpy-24 3’un-translated region (3’UTR) (Plag-2::gfp::dpy-24 3’UTR) 在線蟲DTC中都會持續表現到階段3。我們接下去測試這個調控是在轉譯或是後轉譯層次進行,將Plag-2::dpy-24(cDNA)::unc-54 3’UTR的表現質體微注射到wild-type的線蟲之後發現只要有dpy-24 cDNA表現就可以調控DTC的遷移,因此我們可以知道dpy-24在DTC中的調控層次是在轉譯、後轉譯或是在蛋白質穩定性的層次。 During hermaphrodite development two distal tip cells (DTCs) migrate to generate U-shaped gonadal arms. These cells undergo three phases of migration, they first move along the ventral side to the ends of the body (phase I), then make a dorsal turn (phase II) and finally migrate back to the center of body along dorsal side (phase III). The dpy-24 gene encodes a protein with a PR domain and zinc-finger and likely controls the timing of DTC dorsal migration by transcriptional repression of unc-5. Mutations in dpy-24 resulted in the precocious dorsal turn of DTCs. Immunostaining using anti-DPY-24 antibodies detected DPY-24 protein in DTCs during migration phase I but not II or III. Interestingly, the constitutive expression of dpy-24 partially blocked DTC dorsal turn. These results together indicate that the drop of DPY-24 level is crucial for switching the migration phase of DTCs from I to II. To investigate the cause for the decrease of DPY-24 level in migration phase II, we generated various transcriptional and translational gfp reporter gene fusions. The gfp reporter construct Pdpy-24::gfp::unc-54 3’UTR carrying the dpy-24 promoter and unc-54 3’ un-translational region (UTR) and the plasmid Plag-2::gfp::dpy-24 3’UTR containing the lag-2 promoter and dpy-24 3’ UTR showed GFP expression in all migration phases I, II and III. These results indicate that the drop of DPY-24 level is likely not regulated at the transcriptional level. We then test if the regulation may be at the post-transcriptional, translational or post-translational level. We inject the construct Plag-2::dpy-24(cDNA)::unc-54 3’UTR into N2, and the results show that most of these transgenic animals are wild-type. Therefore, the regulation level of DPY-24 in DTCs is translational level, post-translational level or protein stability level. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/29321 |
全文授權: | 有償授權 |
顯示於系所單位: | 分子與細胞生物學研究所 |
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