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DC 欄位 | 值 | 語言 |
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dc.contributor.author | Ya-wen Chen | en |
dc.contributor.author | 陳雅雯 | zh_TW |
dc.date.accessioned | 2021-07-01T08:12:16Z | - |
dc.date.available | 2021-07-01T08:12:16Z | - |
dc.date.issued | 2001 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75219 | - |
dc.description.abstract | Hedgehog(Hh)是參與多種生物發育過程的分泌性蛋白質,其濃度梯度(concentration gradient)分佈的範圍決定其他型態因數(morphogen)的表現範圍,例如Wingless(Wg)及Decapentaplegic(Dpp)。Hh能分佈的範圍受到膽固醇(cholesterol)修飾及其受體Patched(Ptc)的限制。dispatched(disp)基因是具有母源效應的節向突變基因,其胚胎表皮呈現節向基因(segment polarity gene)典型的lawn-of-denticles性狀;已知Disp參與Hh蛋白質的釋放作用。Disp蛋白質為一膜蛋白(membrane protein)具有兩個巨大的細胞膜外環區(extracellular loop),以及SSD區域(sterol-sensing domain);由脊椎動物的實驗推測SSD可能對膽固醇修飾過的Hh有結合的能力,影響Hh在細胞內的蛋白質傳輸運送過程(protein trafficking)。目前的Hh釋放機制認為原本Hh存在細胞膜上脂肪聚集(lipid raft)之處,經由與Disp蛋白質的SSD區域結合後從細胞膜釋放,傳遞給可能由tout velu(ttv)合成的醣蛋白。醣蛋白運送Hh給前區間細胞,前區間細胞膜上的Ptc受體蛋白質所具有的SSD區域也有與Hh結合的能力,使得Hh又會回到細胞膜上脂肪聚集之處。 我們研究disp的對偶基因(allele):disptr,dispa532,dispa771及加碼射線染色體缺失突變株。dispa532,dispa771及其他缺失突變株皆是由加碼射線(γ-ray)照射產生突變。dispa532,dispa771由南方墨點法得知disp基因附近12.3kb都沒有明顯的缺失存在,其他缺失突變株由第一子代突變搜尋而得,預期可能造成大片段的染色體缺失,仍待進一步的分析。disptr對偶基因是由EMS(ethyl methyl sulfonate)所造成突變株,預期可能產生缺少110胺基酸但具有正確讀碼順序(in frame)的突變蛋白質而造成Disp第一個細胞膜外環區N端的缺失。在disptr中Hh目標基因Ptc蛋白質僅在翅碟(wing imaginal disc)前後區間界線(anterior-posterior compartment boundary)緊鄰的一個細胞中有極微弱的表現,而Hh蛋白質皆堆積在後區間(posterior compartment)細胞;故disptr可能是造成disp基因功能完全缺乏的對偶基因(genetic null allele)。我們推測第一個細胞膜外環區是Disp膜蛋白質能正確分泌Hh蛋白質所需要的;而在disptr突變翅碟中Ptc蛋白質微弱的表現是由前後區間界線上的鄰近兩個細胞直接接觸所造成的結果。 當在翅碟中移除disptr母源與合子的四個對偶基因,Ptc蛋白質的表現量較disptr同源突變的翅碟中的表現量強,因此可見長距離的Hh蛋白質效應(long range effect);亦即遠端disp正常細胞所釋放的Hh蛋白質能夠通過disptr突變細胞群傳送至前端細胞造成目標基因的表現增加。而當後區間內的細胞去除Disp功能時,Hh蛋白質依然可以經過此細胞群,亦即在後區間內的細胞存在負責運送Hh的其他基因。目前僅知Ttv負責前區間細胞Hh的運送,而Ttv是否負責後區間內Hh的運送仍待探究。 | zh_TW |
dc.description.abstract | Secreted Hedgehog (Hh) plays an important role on the developmental process in many organisms. In Drosophila, the concentration gradient of Hh precisely induces the expression of Wingless (Wg) and Decapentapledgic (Dpp) morphogens. Hh undergoes an autocatalytic processing that releases an active 19 kD ligand with cholesterol covalently linked to its C-terminus. The distribution range of Hh is restricted by its receptor, Patched (Ptc), which is also the target gene of hh pathway. dispatched (disp) is required for releasing Hh from the lipid rafts at posterior Hh-producing cells to the anterior Hh-receiving cell. Disp is a membrane protein with 12-transmembrane domain and SSD domain (sterol-sensing domain) and reveals structure homologies with PTC and NPC1 disease genes in vertebrate and their Drosophila homologues. In vertebrates, enzymes involved in cholesterol homeostasis, such as HMG CoA reductase and SCAP (SREBP cleav age-activating protein) have the SSD domain for monitoring sterol levels in membranes. Disp was proposed to interact with cholesterol moiety of Hh via its SSD domain directly, thus might be required for trafficking the cholesterol-modified Hh through the secretory pathway or might displace the cholesterol-modified Hh from the lipid bilayer for effective signaling. We have several alleles of disp: one is EMS-induced disptr, which has a 364 bases deletion around the first intron to the second exon, and two are γ-ray-induced dispa532 and dispa771, both have non-detectable deletions in the 12.3 kb genomic region examined, and others are also γ-ray-irradiation alleles from F1 screening. These alleles could be deficiency cover disp gene waiting for further analysis. In wing imaginal discs, Ptc protein expresses rarely only in the one abutting cell along the anterior-posterior (AP) boundary in homozygous disptr. disptr transcripts may encode truncated but nevertheless in frame protein with 110 amino acids deletion in the first putative extracellular loop. In comparison with the 2-to-3-cell width Ptc staining along the AP boundary in displ(3)S037707 allele in wing discs, we propose that disptr is a functional null allele and the first extracellular loop is functionally required for Hh releasing. After both maternal and zygotic contributions of disptr are removed on wing imaginal discs, Ptc expresses 2 to 3 cells wide in the disptr clone crossing the AP boundary instead of the rare Ptc expression in homozygous disptr. We propose that the rare Ptc expression on the abutting cells in homozygous disptr is not due to the presence of maternal Disp function but the result of direct cell-cell contact along the AP boundary. In addition, the 2 to 3 cell wide Ptc expression in the somatic clones is resulted from the long-range effect of Hh originated from Hh-producing cells. Thus, there must be an unknown factor responsible for transporting the remote wild-type Hh in the posterior compartment trespassing the disptr mutant clones and causing the target gene expression. Ttv-dependent proteoglycan has been reported in transporting Hh in the anterior compartment. In addition to its function in anterior compartment, Ttv-dependent proteoglycan might also be responsible for transportation of Hh in the posterior compartment. | en |
dc.description.provenance | Made available in DSpace on 2021-07-01T08:12:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2001 | en |
dc.description.tableofcontents | 中文摘要…………………………………………………………………………………………1 ABSTRACT…………………………………………………………………………………………3 TABLE OF CONTENT………………………………………………………………………………5 List of tables…………………………………………………………………………………9 List of figures………………………………………………………………………………10 Abbreviations…………………………………………………………………………………12 INTRODUCTION……………………………………………………………………………………14 Development of Drosophila segmentation…………………………………………………14 Segment polarity genes………………………………………………………………………14 Compartment boundary…………………………………………………………………………15 The Hedgehog morphogen………………………………………………………………………16 Vertebrate homologues………………………………………………………………………17 Hedgehog signal transduction………………………………………………………………18 Components on the membrane…………………………………………………………………18 Transducers inside the cell………………………………………………………………19 Lipid modification of Hedgehog……………………………………………………………20 Dispatched releases Hedgehog………………………………………………………………22 Tout velu transports Hedgehog……………………………………………………………22 Patched and Dispatched………………………………………………………………………23 Niemann-PickType C disease…………………………………………………………………24 Sterol-sensing domain………………………………………………………………………24 Release and uptake of Hedgehog……………………………………………………………25 What disp null allele can tell us………………………………………………………26 MATERIALS AND METHODS………………………………………………………………………27 Fly stock and maintenance…………………………………………………………………27 Cuticle preparation…………………………………………………………………………28 The autosomal FLP-DFS technique…………………………………………………………28 Heat shock treatment…………………………………………………………………………29 Neomycin selection……………………………………………………………………………29 Gal4-UAS system………………………………………………………………………………30 Generation of recombinant clones in somatic tissues………………………………30 Fluorescence imaginal discs antibody staining………………………………………31 Fixation and blocking………………………………………………………………………31 Primary antibody………………………………………………………………………………31 Secondary antibody……………………………………………………………………………32 Operation of confocal microscopy…………………………………………………………32 Single fly PCR…………………………………………………………………………………33 PCR examination of dispatched genomic region…………………………………………34 Genomic DNA extraction………………………………………………………………………35 Southern hybridization………………………………………………………………………36 Membrane preparation…………………………………………………………………………36 Probe preparation……………………………………………………………………………36 Hybridization…………………………………………………………………………………37 RNA extraction…………………………………………………………………………………37 cDNA synthesis Reaction……………………………………………………………………38 Antibody preparation…………………………………………………………………………39 Plasmid DNA preparation……………………………………………………………………40 γ-ray treatment………………………………………………………………………………40 Bioinformatics analysis……………………………………………………………………41 RESULT……………………………………………………………………………………………43 Search for chromosome-deficient disp alleles…………………………………………43 Embryonic phenotypes of disp alleles……………………………………………………43 UAS-disp and tublin-disp rescue experiments…………………………………………45 disptr allele can encode a putative deleted protein………………………………46 Domains and motifs search of Disptr protein…………………………………………48 Patched expression pattern in disp alleles……………………………………………48 Hedgehog expression pattern in disp alleles…………………………………………49 The maternal contribution of disptr……………………………………………………51 Southern blotting……………………………………………………………………………52 The second round of γ-ray screening……………………………………………………53 DISCUSSION………………………………………………………………………………………54 disptr is a functional null allele………………………………………………………55 The one Ptc expression cell………………………………………………………………56 displ(3)S037707 is a leaky allele………………………………………………………57 Disp functions in Hh releasing……………………………………………………………58 Hh long-range effect…………………………………………………………………………59 The first extracellular loop of Disp is required for Hh releasing……………60 γ-ray alleles of dispa532 and dispa771………………………………………………62 ACKNOWLEDGEMENT………………………………………………………………………………64 REFERENCES………………………………………………………………………………………65 | |
dc.language.iso | zh-TW | |
dc.title | Dispatched膜蛋白質在第一個膜外蛋白環區的突變影響果蠅Hedgehog分泌蛋白質的釋放 | zh_TW |
dc.title | A mutation in the first extracellular loop of Dispatched transmembrane protein affects the release of Drosophila Hedgehog | en |
dc.date.schoolyear | 89-2 | |
dc.description.degree | 碩士 | |
dc.relation.page | 100 | |
dc.rights.note | 未授權 | |
dc.contributor.author-dept | 生命科學院 | zh_TW |
dc.contributor.author-dept | 動物學研究所 | zh_TW |
顯示於系所單位: | 動物學研究所 |
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