吳益群臺灣大學：分子與細胞生物學研究所何冠緯Ho, Guan-WeiGuan-WeiHo2007-11-252018-07-062007-11-252018-07-062007http://ntur.lib.ntu.edu.tw//handle/246246/49924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.Acknowledgement………………………………………………………...2 Table of Contents………………………………………………………….3 Abstract……………………………………………………………………6 中文摘要…………………………………………………………………...8 Introduction……………………………………………………………….9 Experimental Procedures……………………………………………….12 C. elegans strains…………………………………………………….....12 Vectors and constructs………………………………………………….12 Expression genes constructed by fusion PCR………………………….15 RNA interference assay………………………………………………...17 Transgenic animals assay………………………………………………17 Western blot and immunoflorescent staining……………….………….19 Results……………………………………………………………….…....21 DPY-24 immunostaining show that DPY-24 protein level is down after dorsal turn…………………………………………………………..…..21 Extra-chromosomal array can be used for analyzing DTC migration and gene expression…………………………………………………..…….21 The transcriptional level analyze………………………………………22 The post-transcriptional level analyze…………………………………22 The dpy-24 cDNA only can regulate DTC migration normally……….23 The protein stability analyze…………………………………………...24 Discussion………………………………………………………….……..26 DPY-24 is respond for DTC migration…………………………………26 Extra copy of genes in transgenic lines may cause complex effects...…29 The mRNA of dpy-24 is not inhibit or degraded……………………….31 DPY-24 protein stability is respond for its down-regulation…………...32 References……...…………...……………………………………………33 Tables and Figures………………………………………………….……35 Table. Arrangement of transgenic animals…………………………..…35 Figure 1. Phases of DTC migration…………………………………….36 Figure 2. The florescent marker is expressed in DTC………………….37 Figure 3.The bacteria western of HIS-tagged DPY-24 and GST-tagged DPY-24…………………………...…………………………………….38 Figure 4.The immunostaining of DPY-24 in N2……………………….39 Figure 5.The percentage of GFP-expressed DTC in JK2868, YW446, Plag-2::GFP::dpy-24 3’UTR, and YW562……………………...……….40 Figure 6. The effects of alg-1 RNAi…………………………………...41 Figure 7. The percentage of normal DTC migration phenotypes in YW656, YW657, and YW658 are high……………………………..….42 Figure 8. The effects of ubq-2 RNAi…………………………………...43 Figure 9A.The DTC migration phenotypes in ubq-2 RNAi, smo-1 RNAi, YW659 and a different line generated at the same time with YW659…44 Figure 9B. Arrangement of phenotypes in Figure 8A………………….45 Figure 10.The level of GFP::DPY-24 in DTC drops significantly after dorsal turn………………………………………………………………46 Figure 11 Ratio of percentage of GFP-expressed DTC before dorsal turn and after dorsal turn.…………………………..……………………......47 Figure 12A. Amino acid sequence of DPY-24……...………………….48 Figure 12B.Predicted PEST region of DPY-24 in C.elegans and C. briggssae is conserved…………………………………………………49706063 bytesapplication/pdfen-US線蟲生殖腺發育基因調控distal tip cellgonadogenesisdevelopmentgene regulationprotein stabilityotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/49924/1/ntu-96-R94b43018-1.pdf