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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/26613完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 周子賓(Tze-Bin Chou) | |
| dc.contributor.author | Yi-Chun Liu | en |
| dc.contributor.author | 劉怡君 | zh_TW |
| dc.date.accessioned | 2021-06-08T07:17:31Z | - |
| dc.date.copyright | 2008-08-05 | |
| dc.date.issued | 2008 | |
| dc.date.submitted | 2008-07-25 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/26613 | - |
| dc.description.abstract | 中文摘要
裂解體(Processing bodies，P-bodies)為細胞質中所聚集一群參與5端至3端方向降解mRNA (訊息核糖核酸)之蛋白質。另外，裂解體也包含一些參與抑制mRNA轉譯之蛋白質，監測mRNA品質管制之蛋白質及參與基因沉默之蛋白質。組成裂解體之蛋白質有其同源物存在於酵母菌，果蠅和人類等不同物種之間。在人類細胞中， Hedls (Human enhancer of decapping large subunit)為一裂解體之架構性蛋白質。Hedls能促使hDcp1 (人類去頭蓋蛋白1)及hDcp2 (人類去頭蓋蛋白質2)之結合，且促進hDcp1a對於hDcp2催化去頭蓋之活性。 本篇論文主要探討dHedls對果蠅卵細胞形成期(oogenesis)發育上扮演之角色。經由果蠅跳躍基因(P-element)篩選到一突變對偶基因,dHedlsH159；dHedlsH159同型對偶基因會造成死亡。dHedls突變會造成約12%的胚胎出現部份腹部體節缺失。且知約有15%之 Osk蛋白質擴散而無法正確地貼附在胚胎卵室的後端。 在果蠅S2細胞之細胞質中，dHedls能與果蠅裂解體蛋白之標誌－果蠅去頭蓋蛋白質1 (dDcp1)和果蠅去頭蓋蛋白質2 (dDcp2)座落於同一處；果蠅卵發育過程中(oogenesis)，dHedls能與dDcp1和dDcp2在果蠅卵室內的護理細胞和濾泡細胞座落於同一處，知dHedls為果蠅裂解體之一員。當dHedls突變會減少dDcp1和dDcp2在護理細胞質中表現的數量。反之， dHedls大量表現會增加dDcp1和dDcp2在護理細胞質中表現的數量。dHedls之存在對dDcp1和dDcp2進入至護理細胞質之裂解體扮演相當重要的角色。 另外，dHedls突變呈現多種突變性狀，包含卵巢形態異常，護理細胞入侵卵，及過多護理細胞核存於單一卵室。dHedls基因突變於濾泡細胞會破壞卵表層之細胞骨架：且部份dHedls突變的濾泡細胞會造成細胞不正常之增生。因此知dHedls對維持細胞骨架及正常細胞質分裂扮演重要的角色。 | zh_TW |
| dc.description.abstract | Processing bodies (P-bodies) are specific cytoplasmic foci containing proteins involved in the 5’ to 3’ mRNA decay pathway. Some proteins involved in translational repression, mRNA quality control pathway and RNA-mediated gene silencing together with their mRNA targets are also localized to P-bodies. The components of P-bodies are highly conserved among yeast, human and Drosophila. In human cells, Hedls (Human enhancer of decapping large subunit) is a scaffold protein which interacts with human decapping protein 1 (hDcp1a) and human decapping protein 2 (hDcp2). Hedls promotes dDcp1a to enhance hDcp2 decapping activity.
The aim of this thesis is to analyze the developmental function of Drosophila Hedls, dHedls, in oogenesis. A dHedls strong mutant allele, dHedlsH159, was recovered from P-element excision screen. dHedlsH159 is a homozygous lethal line. 12% of dHedlsH159 GLC embryos displayed mild posterior group embryonic defects. Osk protein was mislocalized in a scattered manner at the posterior of 15% dHedlsH159 GLC oocytes. In S2 cells, dHedls was colocalized with P-bodies markers such as dDcp1 and dDcp2. During oogenesis, dHedls was colocalized with dDcp1 and dDcp2 in nurse cells and follicle cells. The above experiments indicated that dHedls is a component of Drosophila P-bodies. Morever, the numbers of dDcp1 and dDcp2-staining bodies were decreased in dHedls mutant background. Overexpression of dHedls increased the numbers of dDcp1 and dDcp2-staining bodies in nurse cell. Therefore, dHedls plays an important role in targeting dDcp1 and dDcp2 to cytoplasmic P-bodies of nurse cells during oogenesis. Egg chambers of dHedlsH159 GLC (germline clone) displayed pleiotropic phenotypes. First, abnormal ovary morphology was visible. Second, the entire of nurse cell nuclei protruded into oocyte. Third, the multinucleated nurse cells were observed. Cortical actin cytoarchitecture of oocyte was disrupted in follicle cells of dHedlsH159 mutant clones. Some mutant follicle cells proliferated abnormally. We proposed that dHedls maintains actin cytoskeleton and is required for normal cell cytokinesis. | en |
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| dc.description.tableofcontents | 致謝…………………………………………………………………………………i
中文摘要…………………………………………………………………………..ii Abstract……………………………………………………………………………iv Table of Contents……………………………………………..............................vi Table List………………………………………………………………………….xi Table of Figures…………………………………………………………………..xii Primer List………………………………………………………………………..xv Introduction………………………………………………………………………1 1、 The biological role of RNA turnover…………………………………………..1 2、 Two major mRNA degradation pathways in eukaryotes………………………2 2.1、5’ to 3’ mRNA decay pathway…………………………………………...2 2.2、3’ to 5’ mRNA decay pathway……………………………………………3 3、Processing bodies are sites of 5’ to 3’ mRNA degradation and contain factors required for RNA interference and nonsense-mediated decay (NMD) pathways……………………………………………………………….4 4、Dynamic linkage between polysome, P-bodies and stress granules……………7 5、P-bodies exist in different species………………………………………………8 5.1、P-bodies in yeast………………………………………………………….8 5.2、P-bodies in Arabidopsis…………………………………………………..9 5.3、P-bodies in Caenorhabditis elegans………………………………………10 5.4、P-bodies in Drosophila…………………………………………………...10 5.5、P-bodies in human………………………………………………………..11 6、Hedls (Human enhancer of decapping large subunit)/Ge-1 is a component of human processing body……………………………………………………..11 7、To analyze P-bodies in Drosophila during oogenesis…………………………12 8、Overview of Drosophila oogenesis……………………………………………13 9、Dorsal-ventral axis determination……………………………………………...15 10、Anter-posterior axis determination……………………………………………17 10.1、bicoid mRNA localization and expression……………………………..17 10.2、oskar mRNP complex and its posterior localization……………………18 11、Follicle cell differentiation and signaling during oogenesis………………….20 11.1、JAK/STAT signaling pathway………………………………………….21 11.2、SWH / Notch pathways…………………………………………………21 12、Drosophila decapping protein 1, dDcp1, is one component of osk mRNP complex………………………………………………………………………22 13、Drosophila decapping protein 2, dDcp2 is a catalytic decapping enzyme……23 14、The purpose of this thesis……………………………………………………...24 Materials and Methods…………………………………………………………25 1、Drosophila stocks……………………………………………………………..25 2、Germ-line clone generation……………………………………………………25 3、Single fly PCR………………………………………………………………..26 4、Inverse PCR…………………………………………………………………..27 5、Reverse-transcription PCR (RT-PCR)………………………………………..28 6、Cuticle preparation……………………………………………………………29 7、dHedls antibody generation…………………………………………………..29 8、The plasmid construction for over-expressed dHedls…………………………29 9、The plasmid construction for S2 cell staining………………………………..30 10、Whole-mount ovary antibody staining……………………………………….31 11、Micro-injection and transgenic fly……………………………………………31 12、Drosophila S2 cells maintenance……………………………………………32 13、Freezing S2 cells……………………………………………………………..33 14、Thawing S2 cells……………………………………………………………..33 15、Transient transfection of S2 cells…………………………………………….34 16、Fluorescence antibody staining of S2 cells……………………………………35 17、To gather cell lysates for immuno-precipitation (IP) …………………………36 18、Immuno-precipitation…………………………………………………………39 19、Western blot anlaysis…………………………………………………………40 Results……………………………………………………………………………...42 Summary…………………………………………………………………………..42 1、CG6181 is the unique Drosophila Hedls………………………………………45 1.1、Multiple sequences alignment of Hedls protein…………………………45 1.2、dHedls belongs to the β-propeller family of proteins…………………46 1.3、dHedls contains a signal peptide………………………………………..46 2、Created dHedls mutant alleles………………………………………………..47 2.1、BL14124 was the source for local transposition………………………..47 2.2、The manipulation of local transposition………………………………..49 2.3、Recovery of stronger mutant or null allele of dHedls by imprecise excision of dHedlsH83……………………………………………………………..52 2.4、dHedlsH159 is a dHedls strong mutant allele…………………………….53 2.5、Lethal phase of dHedls mutant organism……………………………….54 2.6、The embryos derived from dHedlsH159 GLC females showed abnormal anterior-posterior and dorsal-ventral patterning……………………….54 3、The production of dHedls antibody…………………………………………..58 4、The expression pattern of dHedls in oogenesis……………………………….58 5、dHedls is a component of Drosophila P-bodies……………………………….59 5.1、dHedls was colocalized with dDcp1 and dDcp2 in Drosophila S2 cells..59 5.2、The interaction between dHedls and dDcp1 by immunoprecipitation did not confirm…………………………………………………………………...60 5.3、dHedls was colocalized with dDcp1 and dDcp2 in nurse cells and follicle cells………………………………………………………………………61 5.4、dHedls loss-of-funciton decreased the number of dDcp1 and dDcp2- staining bodies in nurse cell cytoplasm…………………………………..62 5.5、Overexpression of dHedls increased the numbers of dDcp1- and dDcp2-staining bodies in nurse cells……………………………………..63 5.6、Depletion of dDcp2 caused the accumulation and enlargement of dHedls- staining bodies in nurse cells and oocyte………………………………...65 5.7、dHedls was colocalized with dDcp1 and dDcp2 in cellular blastoderm embryos…………………………………………………………………..66 6、Pleitotropic mutant phenotypes were observed in egg chambers derived from dHedlsH159 GLC females………………………………………………………68 6.1、Abnormal ovarian morphology was observed in dHedls mutant background……………………………………………………………...68 6.2、Nurse cell nuclei protruded into oocyte in dHedlsH159 GLC egg chambers………………………………………………………………..69 6.3、Multinucleated nurse cells were observed in dHedlsH159 GLC egg chambers…………………………………………………………………70 7、dHedls functions in follicle cells………………………………………………71 7.1、Follicle cells proliferated in dHedlsH159 GLC egg chambers……………71 7.2、dHedls may affect the cytoarchitecture, actin cortex integrity of oocyte and follicle cell proliferation……………………………………………72 Discussion…………………………………………………………………………75 1、Compare with Hedls protein among different species………………………...75 1.1、Protein structure………………………………………………………..75 1.2、Hedls is a component of P-bodies………………………………………76 1.3、The role of Hedls plays in Dcp1 and Dcp2 interaction ………………..77 1.4、dHedls contains a N-terminal signal peptide……………………………78 2、dHedls is necessary for dDcp1 and dDcp2 targeting to Drosophila P-bodies…79 3、Sequential formation of scaffold proteins in P-bodies…………………………79 4、The dHedls bodies at the posterior pole of stage 2-6 oocyte may be not P-bodies………………………………………………………………………..81 5、dHedls mutant exhibited mild Anterior-posterior defect and mislocalization of Oskar protein…………………………………………………………………..82 6、dHedls mutant exhibited pleiotropic phenotypes………………………………83 6.1、dHedls mutant exhibited multinucleated nurse cells………………….84 6.2、The nuclei of nurse cell protruded into developing oocyte……………86 6.3、dHedls mutant exhibited abnormal ovary morphology……………….88 6.4、dHedls mutant exhibited follicle cell overgrowth…………………….89 6.5、dHedls mutant exhibited disrupted actin cytoskeleton………………..91 References………………………………………………………………………..94 Table List Table 1、The embryonic mutant phenotypes derived from BL14124, dHedlsH83 and dHedlsH159 GLC females and percentages of each phenotype were shown. List of Figures Fig.1、General mRNA decay pathway and basic components of P-body (Processing body) Fig.2、A model is linking RNAi and GW body (Processing body or Dcp1 body) assembly and function Fig.3、A model is for the cycling of eukaryotic mRNA between different subcellular components. Fig.4、Formation and development of Drosophila egg chamber. Fig.5、Follicle cell differentiation and signaling during Drosophila oogenesis Fig.6、The genomic map of dHedls and multiple sequences alignment of N-terminal region of dHedls with its homologues were identified by BLAST search. . Fig.7、Schematic illustrations present Hedls proteins among different species and WD40 domain Fig.8、dHedls contains a signal peptide at N-terminal region Fig.9、The manipulation of P-element local transposition was shown and H83 insertion line was obtained Fig.10、The insertion site of dHedlsH83 and RT-PCR products for dHedls transcripts were shown Fig.11、Cuticle preparation of unhatched embryos derived from dHedls mutant females Fig.12、Schemes of dHedlsH83 local transposition screen and insertion site were shown Fig.13、PCR analysis and the deletion region in dHedlsH159 mutant line were shown Fig.14、dHedls transcripts were amplified by RT-PCR Fig.15、Cuticle preparation of unhanched embryos derived from dHedlsH159 mutant GLC females. Fig.16、dHedls mutant affected the localization of Osk in stage 9-10 egg chambers Fig.17、The abnormal mutant phenotypes of dorsal appendage were observed in dHedlsH159 GLC embryos. Fig.18、Grk distribution was unaffected in dHedlsH159 GLC oocyte Fig.19、Distribution of dHedls was detected in germarium and S2-S10 follicles Fig.20、The distribution of dHedls in stage 10 egg chambers was shown Fig.21、In Drosophila S2 cells, dHedls was colocalized with dDcp1 and dDcp2 Fig.22、Immunoprecipitation was used to examine the interaction between dDcp1 and dHedls Fig.23、dHedls was colocalized with dDcp1 and dDcp2 in egg chambers of wild type Fig.24、dHedls signal was decreased in dHedlsH159 GLC mutant background Fig.25、dHedls mutant reduced the cytoplasmic dDcp1-staining bodies in nurse cells Fig.26、dHedls mutant led to the reduction of cytoplasmic dDcp2-staining bodies in nurse cells Fig.27、Overexpression of dHedls increased the number of dDcp1 and dDcp2-staining bodies in nurse cell cytoplasm Fig.28、dDcp2 mutant caused the accumulation and enlargement of dHedls-staining bodies. Fig.29、dHedls formed discrete cytoplasmic particles in cellular blastoderm embryos and were colocalized with dDcp1 and dDcp2 Fig.30、Abnormal morphology of ovaries were found in the dHedlsH159 mutant background Fig.31、The aggression of nurse cell nuclei protruded into oocyte of dHedlsH159 GLC egg chambers Fig.32、dHedlsH159 GLC egg chambers exhibited mild defect of cytokinesis Fig.33、dHedlsH159 GLC egg chambers exhibited abnormal proliferation of follicle cells Fig.34、Mosaic analysis of dHedls function in follicle cells. | |
| dc.language.iso | en | |
| dc.subject | 細胞增生 | zh_TW |
| dc.subject | 裂解體 | zh_TW |
| dc.subject | 卵生成 | zh_TW |
| dc.subject | 訊息核糖核酸分解 | zh_TW |
| dc.subject | P-bodies | en |
| dc.subject | oogenesis | en |
| dc.subject | cell proliferation | en |
| dc.subject | mRNA degradation | en |
| dc.title | 果蠅促進去頭蓋大單元蛋白(dHedls)之發育遺傳分析 | zh_TW |
| dc.title | Developmental genetic study of Drosophila homologue of Human enhancer of decapping large subunit, dHedls | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 96-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蘇銘燦(Ming-Tsan Su),董桂書(Kuei-Shu Tung),李士傑(Shyh-Jye Lee) | |
| dc.subject.keyword | 裂解體,卵生成,訊息核糖核酸分解,細胞增生, | zh_TW |
| dc.subject.keyword | P-bodies,oogenesis,mRNA degradation,cell proliferation, | en |
| dc.relation.page | 178 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2008-07-28 | |
| 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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