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研究生:張雅惠
研究生(外文):Ya-Hui Chang
論文名稱:在早期胰臟發育中特定表現的Rbms3之特性分析
論文名稱(外文):Characterization of Rbms3(RNA binding motif, single strand interacting protein 3), a novel protein that is preferentially expressed in early developing pancreas
指導教授:江明格
指導教授(外文):Ming-Ko Chiang
學位類別:碩士
校院名稱:國立中正大學
系所名稱:分子生物研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:英文
論文頁數:64
中文關鍵詞:在早期胰臟發育中特定表現的Rbms3之特性分析
外文關鍵詞:pancreas developmentsingle strand interacting protein 3)Rbms3(RNA binding motif
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核酸結合區單股作用蛋白3 (RNA binding motif, single strand interacting protein 3-RBMS3)是一個經由南西方分析法審查人類纖維母細胞基因庫中,鑑定出一個新的基因產物。根據RBMS3 cDNA 的序列判斷,其蛋白質由414個胺基酸組成並含有兩對核酸結合區(RNA binding motif; figure 16)。RBMS3 結構非常接近於c-myc 基因單股結合蛋白(c-myc gene single-strand binding protein: MSSPs)此基因是藉著與C-MYC蛋白作用進而調控去氧核酸的複製,轉錄,凋亡及細胞週期過程。ㄧ般而言,會與核酸結合的蛋白質往往參與釵h細胞的加工;這樣的情況常決定生物的生存與否。
我們發現到RBMS3在老鼠體內有不同的表現型態,另外針對老鼠中胰臟各細胞所作的基因晶片分析也得到RBMS3表現的痕跡。為了更加了解RBMS3在胰臟的發育上有何影響,使用了釵h實驗去驗證;例如定位雜合法(In situ hybridization)、免疫螢光染色法(Immunofluorescence) ,從這些實驗觀察到Rbms3的蛋白質及RNA在胚胎胰臟中有特異性的表現。另外西方點墨法(Western blot)也偵測到Rbms3蛋白質在老鼠胚胎表現的訊號,但Rbms3在成鼠胰臟中的表現並無法確認;因為北方點墨法(Northern blot)並沒有偵測到Rbms3 RNA的訊號。而利用酵母菌雙雜合法(Yeast two hybrid)去觀察RBMS3與其他蛋白質是否有相互作用的關係,結果發現到其中有一個候選基因-SPIN3 (spindlin family, member 3);此基因是對於發育上似乎也有一些弁遄A於是若能以CO-immunoprecipitation或 Pull down assay的實驗證明其相互作用之關係後,未來希望能藉此發現RBMS3對胰臟發育上的調控以期待能對於胰臟疾病有進ㄧ步的治療及控制。
Southwestern screening of human fibroblast cDNAs library has to the identification of a novel gene product, RBMS3(RNA binding motif, single strand interacting protein 3). RBMS3 gene encodes a protein of 414 amino acid which contains two pairs of RNA binding motifs . It is closely related to MSSPs(
c-myc gene single-strand binding proteins) . MSSPs are believed to regulate DNA replication, transcription, apoptosis and cell cycle progression by interacting with the C-MYC protein. Proteins with RNA –binding motifs involve in a variety of cellular processes that are critical to the survival of the organism.
Previously, expression of RBMS3 has been detected specifically in mouse embryonic pancreas with the use of microarray assay. To understand the effect of Rbms3 during pancreatic development, in situ hybridization and immunofluorecsence staining were applied to confirm the presence of RBMS3 in the embryonic pancreas. And western blot assay was also detected Rbms3 signal in mouse embryos but the adult mouse pancreas protein lysate was not made sure to detect Rbms3 signal. Because this was no Rbms3 RNA signal in adult mouse pancreas total RNA. In addition, in order to explore the functions of RBMS3, a yeast two-hybrid screening was performed in order to identified proteins which associated with RBMS3. The one of candidates gene-SPIN3(Spindlin family, member 3) which was relate to development was researched. To confirm the interaction, co-immunoprecipitation and pull down assay were used. The co- immunoprecipitation still was not observed the interaction of Rbsm3 and SPIN3, but pull down assay will be used to direct interaction of Rbms3 and SPIN3. Make use of understanding the roles and functions of Rbms3 during pancreas development, we hope to provide some contributes in pancreatic diseases.
Abstract(Chinese version)……………………………………………………… 3
Abstract(English version)……………………………………………………… 4
Chapter 1 Introduction and background……………………………………… 7
1.1 Introduction to pancreatic structure………………………………………….. 7
1.2 Exocrine………………………………………………………………………. 8
1.3 Endocrine……………………………………………………………………… 9
1.4 Function of the pancreas……………………………………………………..... 10
1.5 Pancreas forming………………………………………………………………. 11
1.6 Identification of RNA binding motif, single strand interacting protein 3
(RBMS3)……………………………………………………………………….. 12
Chapter 2 materials and methods………………………………………….......... 14
2.1 Gene expression construct preparation………………………………………… 14
Reverse transcriptase polymerase chain reaction (RT-PCR)…………………... 14
Polymerase chain reaction(PCR)……………………………………………… 14
The insert and vector ligation………………………………………………….. 15
2.2 In situ hybridization…………………………………………………………… 16
In vitro transcription…………………………………………………………… 16
non-radioactive In situ hybridization to tissue sections……………………….. 16
2.3 Total RNA isolation…………………………………………………………… 19
2.4 Northern blot…………………………………………………………………... 20 Agarose/Formaldehyde Gel Electrophoresis………………………………….. 20
Transfer of RNA from Gel to Membrane……………………………………... 20
Hybridization analysis………………………………………………………… 20
2.5 Protein extraction……………………………………………………………… 22
Purify Rbms3 serum antibody by CYANOGEN BROMIDE ACTIVATED MATRICES (SIGMA)………………………………………………………… 22
2.6 Western blot…………………………………………………………………..... 23
2.7 Coomassie blue staining……………………………………………………….. 24
2.8 Yeast two hybrid……………………………………………………………….. 24
Principle of the two-hybrid assay……………………………………………… 24
Transform plamid into yeast…………………………………………………… 25
Check 3-amino-1,2,4-triazole (3-AT) concentration…………………………... 25
Two-Hybrid Library Screening Using Yeast Mating………………………….. 25
Yeast plasmid isolation………………………………………………………… 26
β-galactosidase assays…………………………………………………………. 27

2.9 Cell culture and transfection…………………………………………………… 28
Cell culture…………………………………………………………………… 28
Transfection……………………………………………………………………..28
2.91 Immunostaining………………………………………………………………. 28
2.92 Co-immunoprecipitation………………………………………………………29
2.93 Fixation and Embedding……………………………………………………….29
Chapter 3 Result……………………………………………………………………30
3.1 Production of anti-Rbms3 antibody………………………………………..30
3.2 Expression of Rbms3 in adult mouse tissues and embryos……………… 31
3.3 Identification of Rbms3-interacting proteins………………………………32
Chapter 4 Discussion……………………………………………………………….35
Chapter 5 References………………………………………………………………39
Chapter 6 Figures and table……………………………………………………….42
Appendix A………………………………………………………………………….62
Appendix B………………………………………………………………………….64
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