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研究生:林忠德
研究生(外文):Chung-De Lin
論文名稱:探討moph基因在果蠅發育上的分子機制
論文名稱(外文):Characterization of the molecular function of moph in Drosophila development
指導教授:皮海薇
指導教授(外文):Hai-Wei Pi
學位類別:碩士
校院名稱:長庚大學
系所名稱:基礎醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:49
中文關鍵詞:翅脈果蠅發育
外文關鍵詞:mophveinEGFR
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過去實驗室用EP 篩選出一個全新的基因,稱之為moph (modifier of phyllopod)。moph在果蠅物種的序列比較中有高度的相似性。moph 可轉錄出1.5Kb的mRNA且有一個小的ORF(49a.a.)包含在其中。為了去分析moph在分子層次上機制, 我們製作了數種UAS-moph 的基因轉殖果蠅。我們發現大量同時表達moph 以及phyllopod 可以加成性的造成果蠅剛毛異常生長,但是僅表達小的ORF是不足以造成剛毛異常生長的現象。另外將moph ORF的start codon 從ATG突變成TAG也不會影響整個moph基因造成剛毛異常生長的能力。藉由大量表達moph的這些實驗,我們推測moph是透過整段的RNA來執行功能。
利用northern blot發現moph表達的時期在果蠅的胚胎,幼蟲,蛹期,以及成蟲。透過 phyllopod 1.8Kb mRNA當作標準,發現moph 的mRNA長度稍微低於1.8Kb。In situ hybridization 顯示moph 的表達位置跟EGFR 有相關性。為了了解moph是否受到EGFR 訊號的調控, 將EGFR negative regulator cbl 大量表達在wing disc。根據這個實驗我們發現,在果蠅翅脈上的moph 訊號是經由EGFR活性所調控。
為了找出moph的mutant phenotype,我們製作了moph dsRNA基因轉殖果蠅。northern blot 以及RT都確定dsRNA的果蠅可以有效調降moph的表達。利用Dpp-Gal4專一性的在wing disc A/P boundary調降moph表現量,藉此來測試moph mutant phenotype。moph 表現量下降所造成翅脈上的phenotype類似EGFR活性輕微上升所造成的phenotype。為了測試EGFR與moph的genetic interaction, 在moph mutant 之下大量表現EGFR 的constitutively actived form (λTop)。在一個copy的moph mutant 之下可以輕微促進大量表達λTop所造成在翅脈上的phenotype。根據這些結果,我們推測,moph 在翅脈發育中能調降EGFR的活性。
Previous EP screening in our lab found a novel gene moph (modifier of phyllopod). Sequence comparison showed moph was highly conserved in Drosophila species. moph encodes a 1.5kb putative non-coding RNA with a small predicted ORF (49 a.a.). In order to analyze moph molecular functions, several UAS-moph constructs were generated. While overexpression of the 1.5kb full-length moph together with phyl could promote ectopic bristle formation, no ectopic ES organs were found when only the putative polypeptide was overexpressed. Furthermore, change of the start codon of the moph putative ORF to TAG didn’t affect the ability of moph to promote ectopic ES organs. Thus, these overexpression assays suggested that moph functions as a non-coding RNA.
Northern blot result showed moph is expressed in embryo, larva, pupal, and adult. The length of the moph mRNA was slightly shorter than that of the phyllopod 1.8Kb mRNA. moph in situ hybridization data showed moph expression pattern was correlated with EGFR signaling. To test whether moph was regulated by EGFR signaling, EGFR negative regulator cbl was overexpressed in wing disc. We found moph expression was dependent on EGFR activity in wing vein, suggesting that moph expressionis activated by EGFR signaling activity.
To find out the moph mutant phenotypes, moph dsRNA transgenic fly was generated. Both northern and RT-PCR analyses confirmed that moph expresssion was indeed down-regulated by this dsRNA. To examine moph mutant phenotypes by RNA interference, Dpp-Gal4 was used to knockdown moph in A/P boundary of wing disc. moph knockdown phenotype was similar to that of the weak EGFR activated phenotype in wing vein. Genetic interaction between EGFR and moph was tested by overexpression of the constitutively active form of EGFR (λTop) in moph heterozygous mutant background. Vein-thickening phenotype caused by λTop overexpression could be mildly enhanced by loss of one copy of moph. Based on these results, we speculate that moph negatively regulate EGFR signaling activity during wing vein development.
<Table of Contents>
指導教授推薦書
口試委員會審定書
授權書………………………………………………………………… iii
誌謝…………………………………………………………………… iv
Abstract (Chinese)…………………………………………………… v
Abstract………………………………………….………………….….vii
Table of contents……………………………………............................. ix
Chapter I Introduction…………………………………….…………1
Chapter II Material and Methods …………………………………..5
2.1 Fly strains……………………………………..………………5
2.2 Molecular cloning……………………………………………5
2.3 Drosophila total RNA extraction……………………………7
2.4 Northern Blot…………………………………………………7
2.5 In situ hybridization…………………………………………8
2.6 5’RACE………………………………………………………9
Chapter III Result……………………………………………………11
3.1 moph sequence comparison between three different Drosophila species……………………………………………..11
3.2 moph overexpression phenotype is similar to EPC05-660…11
3.3 moph functions as a non-coding RNA………...…………….12
3.4 Prediction of moph RNA secondary structure…..…………13
3.5 moph activity wasn’t affected in one copy of dicer1 or
dicer2 mutant background………..………………………….14
3.6 The expression of moph through all the Drosophila
life cycle………………………………………………………15
3.7 moph overexpression phenotype in Drosophila wing…...… 15
3.8 moph expression pattern in developing wing………..……16
3.9 Generation of UAS-dsmoph………………………………..16
3.10 UAS-dsmoph could cause ectopic vein in more sensitive background………………………………………….…….. 18
3.11 Genetic interaction of moph and EGFR pathway……….. 18
3.12 Expression of moph could be activated by EGFR
Signaling……………………………………………..…...... 19
3.13 Amplication of the 5’ cDNA end of moph………...……… 20
Chapter IV Discussion
4.1 moph mutant phenotype is hard to identify………...………21
4.2 Molecular function of moph…………………….…..……….22
4.3 The small open reading frame of moph……………....……..22

Referance……………………………………………………………….24

Figures and Tables…………………………………………………..30
1. Akira Nakamura, Reiko Amikura, Masanori Mukai, Satoru Kobayashi, Paul F. Lasko. "Requirement for a Noncoding RNA in Drosophila Polar Granules for Germ Cell Establishment", Science, 274, pp. 2075-2079, December 1996.

2. Rui Gonc¸ alo Martinho, Prabhat S. Kunwar,Jordi Casanova,2and Ruth Lehmann. "A Noncoding RNA Is Required for the Repression of RNApolII-Dependent Transcription in Primordial Germ Cells", Current Biology, 14, pp. 159-165, January 2004.

3. Kirsten E. Hardiman,1Rachel Brewster, Shaema M. Khan, Monika Deo and Rolf Bodmer. "The bereft Gene, a Potential Target of the Neural Selector Gene cut, Contributes to Bristle Morphogenesis", Genetics, 161, pp. 231-247, May 2002.

4. Rainer B. Lanz, Neil J. McKenna, Sergio A. Onate, Urs Albrecht, Jiemin Wong, Sophia Y. Tsai, Ming-Jer Tsai, and Bert W. O’Malley. "A Steroid Receptor Coactivator, SRA, Functions as an RNA and Is Present in an SRC-1 Complex" Cell, 97, pp. 17–27, April 1999.

5. A. T. Willingham,A. P. Orth, S. Batalov, E. C. Peters, B. G. Wen, P. Aza-Blanc, J. B. Hogenesch. P. G. Schultz. "A Strategy for Probing the Function of Noncoding RNAs Finds a Repressor of NFAT", Science, 309, pp. 1570-1573, September 2005.

6. Ben-Zion Shilo. "Signaling by the Drosophila epidermal growth factor receptor pathway during development", Experimental Cell Research, 284, pp. 140–149, March 2003.

7. Fernando J. Diaz-Benjumea and Ernst Hafen. "The sevenless signalling cassette mediates Drosophila EGF receptor function during epidermal development", Development, 120, pp. 569-578, 1994.

8. Annabel Guichard, Brian Biehs, Mark A. Sturtevant1, Laura Wickline1, Julie Chacko, Katherine Howard and Ethan Bier. "rhomboid and Star interact synergistically to promote EGFR/MAPK signaling during Drosophila wing vein development", Development, 126, pp. 2663-2676, 1999.

9. Peleg Hasson, Nitir Egoz, Clint Winkler, Gloria Volohonsky, Songtao Jia, Tama Dinur, Talila Volk, Albert J Courey & Ze’ev Paroush. " EGFR signaling attenuates Groucho-dependent repression to antagonize Notch transcriptional output", Nature Genetics, 37, pp. 101-105, January 2005.

10. Jose F. de Celis1, Sarah Bray and Antonio Garcia-Bellido. "Notch signalling regulates veinlet expression and establishes boundaries between veins and interveins in the Drosophila wing", Development, 124, pp. 1919-1928, 1998.
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