周子賓臺灣大學：分子與細胞生物學研究所陳昭翰Chen, Chao-HanChao-HanChen2007-11-252018-07-062007-11-252018-07-062006http://ntur.lib.ntu.edu.tw//handle/246246/49886在酵母和人類的系統中,去頭蓋蛋白已被充分研究。研究發現Dcp1會和Dcp2 形成 heterodimer, 此酵素複合體具有去除訊息RNA頭蓋之活性。Dcp2是去頭蓋作用的催化中心, 且Dcp1被認為能增進其活性。另外, 越來越多的證據顯示許多蛋白質亦參與在此複合體中。Dcp1 和 Dcp2 的另一個特徵是他們會與 Xrn1 , Edc3 和 Dhh1 這些參與在訊息RNA降解路徑上的成員共同在細胞質內形成聚集點, 這個特殊的結構被稱作 P body ,且被認為是訊息 RNA 被進行降解的地點。 我們的之前的研究發現果蠅去頭蓋蛋白 1, dDcp1, 是 oskar mRNP的一個成員且和其定位於卵後端有關 (Lin et al., 2006)。 dDcp1突變時會造成果蠅胚胎腹節發育缺失, 此外, 其他 oskar mRNP 的成員的正確定位亦受到破壞, 如Exu , Yps 以及Orb. 這個發現揭示了訊息RNA在定位 ,轉錄, 轉譯和降解中間的緊密連接的可能性。 經過果蠅基因體比對, 我們找到了唯一可能的 dDcp2 基因, CG6169 。我們分析 dDcp2 突變對偶基因, BG1766, dDcp2de21,發現兩個突變對偶基因都造成前後及背腹的發育缺失。而這些特殊的性狀可能是 Osk, Stau 和 Vasa 蛋白沒能正確座落至卵後端所致。 另一個有趣的發現是當 dDcp2 基因被刪除時, 果蠅卵室內之護理細胞 (nurse cell)內, 可能的 P body 結構會發生明顯的放大和累積, 這也許是訊息RNA 降解路徑受阻的結果。Decapping complex in yeast and human systems are well studied. It is reported that Dcp1 and Dcp2 form a heterodimer with decapping activity. Dcp2 is the catalytic center of decapping process and Dcp1 is believed to promote the decapping activity. And increasing lines of evidence show that many proteins may be associated in the decapping complex. Another distinct feature of Dcp1 and Dcp2 is their colocalization within cytoplasmic foci associated with other mRNA degradation components such as Xrn1, Edc3 and Dhh1. This specialized structure is called processing body (P body) which is referred to as the sites for mRNA degradation. Our previous research uncovered Drosophila decapping protein 1,dDcp1, is a novel component of oskar mRNP complex and directs its posterior localization in the oocyte. (Lin et al., 2006b) dDcp1 mutant causes posterior group phenotype. And dDcp1 is also required for the proper posterior localization of other oskar mRNP complex component, such as Exu, Yps, and Orb. This discovery reveals the possibility of close linkage among transportation, transcription, translation and degradation. According to the Drosophila genome wide gene BLAST result, we uncovered the putative and unique dDcp2 gene, CG6169. We analyzed the dDcp2 mutant allele, BG1766, and dDcp2 null allele, dDcp2de21. Both mutant alleles cause anterior- posterior and dorsal-ventral patterning defect. And the distinct phenotype could be the consequence of mislocalization of Osk, Stau and Vasa proteins. The other interesting finding is that deletion of dDcp2 gene causes the enlargement and accumulation of P body-like structure, which may be the result of the mRNA decay pathway deficiency.中文摘要 1 Abstract 2 Table of Content 3 List of abbreviations 9 List of primers 10 Introduction 11 Drosophila oogenesis 11 Microtubule network polarity during oogenesis 12 Anterior- posterior axis determination 13 bicoid mRNA localization and expression 15 Regulation of oskar mRNA localization and expression 15 oskar mRNA localization 15 oskar mRNA expression 17 Vasa localization 18 Dorsal-ventral axis determination 10 Transcript Stability During oogenesis 21 mRNA turnover pathways 22 General mRNA decay pathways 22 Specialized mRNA decay pathways 24 Present study of Dcp1 and Dcp2 25 dDcp1 is a novel posterior group gene 27 The Drosophila decapping protein 2, dDcp2 28 About the thesis 29 Material and methods 30 Drosophila stocks 30 FLP-DFS technique 30 Local transposition screen for dDcp2 mutant allele 31 Creation of dDcp2 null allele by imprecise excision 32 Germ-line clone generation 32 Cuticle preparation 32 Immunofluorescence for ovary 33 Single fly PCR 34 Inverse PCR 34 Western blot analysis 35 Repetitive epitode cloning 35 Protein expression and purification 36 Protein expression 36 Protein purification by 6xHis binding resin 37 RT-PCR 38 Results 39 CG6169 is the unique Dcp2 homoloque in Drosophila 40 dDcp2 mutant allele, BG1766, cause posterior phenotype and interfere Dorsal-ventral patterning 41 Introduction of BG1766 41 Phenotypes of BG1766 allele 42 P element local tranposition screen for hypomorph dDcp2 allele 42 Sequence analysis of dDcp2 alleles 44 BG1766 44 BG325 44 BG315 45 BG21 45 Creation the dDcp2 null allele, dDcp2de21 46 Thestrategy 46 Procedures of the excision screen 46 The result of the BG21 deletion screen 47 dDcp2de21 chromosome sequence analysis 47 The deletion region of the deletion lines 47 The residual fragment between the break points 48 dDcp2de21 is a dDcp2 null allele 48 Lethal phase of dDcp2 mutant organism 49 dDcp2 null allele, dDcp2de21 also cause the abnormal A-P and D-V patterning 50 A-P patterning defects 50 D-V patterning defects 51 Other unusual phenomenon observed in dDcp2de21 germ line clone 51 dDcp2de21 allele specifically disrupts dDcp2 gene function 51 dDcp1 and dDcp2 mutant alleles share similar embryonic phenotype 52 dDcp2 mutant alleles cause mislocalization of maternal product 53 Osk 53 Stau 53 Vasa 54 Deletion of dDcp2 cause the accumulation and enlargement of putative P body structures 55 Discussion 56 dDcp2de21 allele is a dDcp2 null allele and impairs dDcp2 gene function without disrupting the neighboring diablo gene function 56 dDcp2 gene disruption screen 57 The efficiency of dual P-element excision 57 Application of BG325 allele 58 The Dumping defect in dDcp2de21 GLC egg chambers 59 Posterior phenotype presented in dDcp2 mutant may independent of its decapping function 60 dDcp2 may involved in actin organization 61 dDcp2 mutant cause A-P and D-V patterning defect and mislocalization of maternalproducts 62 dDcp2de21 does not affect Vasa-containing structures in early stage egg chambers 63 dDcp1-containing structures are accumulated and enlarged in dDcp2de21 GLC egg chambers 64 The complexity of the distribution of distinct foci in nurse cells 65 The hypothesis 67 List of Tables Table 1. The ratio of different posterior phenotype of embryos derived from BG1766 and dDcp2de21 germ line clone females Table 2. The ratio of different dorsal appendage fusion phenotype of embryos derived from BG1766 and dDcp2de21 germ line clone females Table 3. Osk, Vas protein localization in the dDcp2de21 GLC oocytes Table 4. Stau protein localization in the dDcp2de21 GLC oocytes List of Figures Fig.1 Asymmetric distribution of maternal determinants for axes formation Fig.2 mRNA decay pathway Fig.3 The major concept of DFS-FLP technique for germ line clone generation Fig. 4 The alternative spicing of dDcp2 isoforms Fig.5 Insertion sites of dDcp2 alleles, BG1766, BG325, BG315 and BG21 Fig.6 The mechanism of mutagenesis by pGT1 insertion line Fig.7 The RT-PCR products for aberrant dDcp2 transcripts in BG1766 insertion Line Fig.8 Schemes of BG1766 local transposition screen and BG21 imprecise excision screen Fig.9 Entire dDcp2 coding sequence is deleted in dDcp2de21 allele Fig.10 The RT-PCR products for dDcp2 transcripts Fig.11 Anti-dDcp2 antibody immunostaining of WT and dDcp2de21 GLC ovaries Fig.12 The map shows two dDcp2 isoforms and two genome fragments for complementary tests Fig.13 The RT-PCR products for diablo transcript Fig.14 Cuticle preparation of unhatched embryos Fig.15 Dorsal appendage fusion phenotype and dumping defect is also found in dDcp1 mutant Fig. 16 dDcp2de21 mutant affects the localization of Osk in stage 8-10 egg chamber Fig. 17 The localization of Stau protein on dDcp2de21 mutant background Fig. 18 The Vasa protein expression pattern is disrupted when dDcp2 is mutated Fig.19 The Vasa antibody staining for dDcp2de21 early stage egg chambers are resemble to WT situation Fig. 20 Abnormal actin particles exist in dDcp2de21 GLC ooctytes Fig.21 Abnormal P body-like structures are accumulate when dDcp2 is mutated Fig.22 The possible indirect effects of dDcp1 and dDcp2 in A-P pattering Fig. 23 The model: aberrant P body disrupt osk mRNP assembly and transposition Acknowledgments 93 Reference 942658300 bytesapplication/pdfen-US果蠅去頭蓋蛋白果蠅卵發育dDcp2Drosophila oogenesisotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/49886/1/ntu-95-R93b43020-1.pdf