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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 莊寧寧 | |
| dc.contributor.author | Han-Yi Fu | en |
| dc.contributor.author | 傅瀚儀 | zh_TW |
| dc.date.accessioned | 2021-06-13T02:06:04Z | - |
| dc.date.available | 2007-07-24 | |
| dc.date.copyright | 2007-07-24 | |
| dc.date.issued | 2007 | |
| dc.date.submitted | 2007-06-30 | |
| dc.identifier.citation | Anderson, R. G. W. (1998). The caveolae membrane system. Annual Review of Biochemistry 67, 199-225.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/30515 | - |
| dc.description.abstract | E-Ras主要表現在未分化的胚胎幹細胞中，跟一般的Ras蛋白質序列相比，E-Ras蛋白質本身即具備致癌Ras的蛋白質突變胺基酸，可維持GTP-locked，並在N端有一段多餘的序列。E-Ras主要是以GTP結合的形式使細胞轉型。本研究建立了E-ras穩定轉染的BALB/3T3纖維母細胞株，並探討caveolin-1在E-ras轉染細胞中所扮演的功能。利用Triton X-114分萃膜蛋白後，發現caveolin-1只分布在疏水層，而在轉型細胞中其表現則較少；caveolin-2表現量則持平，並有些分佈到親水層；此外，有很多RasGAP集中在Triton X-114親水層。利用蔗糖梯度超高速離心法處理後，發現caveolae的密度在E-ras轉染的細胞中會增加。這種現象可以直接利用大腸桿菌表現的重組E-Ras及KB-Ras蛋白質於in vitro實驗中再現。因此，我們提出E-Ras和KB-Ras可直接經由細胞質傳送到caveolae中，並造成caveolae密度增加。 | zh_TW |
| dc.description.abstract | E-Ras is expressed in undifferentiated embryonic stem (ES) cells. E-Ras proteins have mutation sites responsible for trapping GTP as oncogenic Ras, plus an extra N-terminal sequences which is not found in conserved Ras protein sequences. E-Ras proteins are mainly GTP-bounded and easily lead to cellular transformation. We have established stable clones of BALB/3T3 cells transfected with E-ras and used this cell line to study the functional role of the caveolin-1 upon cells transfected with E-ras. Using Triton X-114 to fractionate membrane proteins, caveolin-1 was exclusively clustered in Triton X-114-B (hydrophobic phase), with decreased amounts in cells transformed with E-ras, whereas caveolin-2 exhibited no difference of amounts in both Triton X-114-H (hydrophilic phase) and Triton X-114-B. Furthermore, RasGAP, the GTPase activator protein occurred mainly in Triton X-114-H. The expression of RasGAP was significant in Triton X-114-H upon transformation with E-ras. Of interest, caveolae was founded with heavier density upon transformation with E-ras as revealed wtih sucrose gradient ultracentrifugation. This transition of density in caveolae could be reconstituted with recombinant E-Ras proteins. The finding was confirmed with recombinant KB-Ras(Q61K) proteins in cells without transformation. Therefore, E-Ras and KB-Ras(Q61K) may share a similar route from cytosol to caveolae. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T02:06:04Z (GMT). No. of bitstreams: 1 ntu-96-R94b41001-1.pdf: 1303941 bytes, checksum: ce283869c26e6a6c276718cfd768dcee (MD5) Previous issue date: 2007 | en |
| dc.description.tableofcontents | 口試委員會審定書 I
誌謝 II 中文摘要 III Abstract IV Table of contents VI Index of figures VIII Introduction 1 General properties of Ras protein 1 Tertiary structure and conserved nucleotide binding motif of Ras protein 1 C-terminus of Ras protein for membrane localization and transformation 3 Characteristics of E-Ras 5 Regulatory role of RasGAP 6 General properties of caveolins 7 Caveolins and cell transformation 8 Materials and Methods 10 Reagents 10 Cell culture 11 Generation of E-ras constructs 12 Transfection of E-ras 12 Identification of E-Ras protein expression by in vivo staining of tetracysteine tag 13 Anchorage-independent growth assay 14 Sucrose gradient ultracentrifugation 14 Immunfluorescence microscopy of caveolin-1 15 Membrane isolation 16 Phase separation of membrane pellet proteins with Triton X-114 16 Immunoprecipitaion of caveolin-1, PY27-caveolin-2 or RasGAP from Triton-X 114 phase fractions 17 Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) 18 Western immunoblotting analysis 18 Results 20 Expression of E-Ras proteins in BALB/3T3 cells transfected with E-ras 20 Enhanced anchorage-independent growth in cells transfected with E-ras 20 Downregulation of caveolin-1 and overexpression of RasGAP upon cells transfected with E-ras 21 Redistribution of caveolae to higher sucrose density in cells transfected with E-ras 22 Location of caveolin-2 in sucrose gradient in cells transfected with E-ras 22 Location and pattern of caveolin-1 in cells transfected with E-ras 23 Incorporation of Ras proteins into caveolae 23 Recruitment of caveolin-2 with RasGAP 24 Discussion 25 Transformation of cells transfected with E-ras 25 Different expression pattern of caveolin-1 and caveolin-2 upon cells transfected with E-ras 25 Redistribution of caveolae to heavier density upon cells transfected with E-ras 26 Incorporation of E-Ras to caveolae 27 Interaction with RasGAP and caveolin-2 28 References 30 | |
| dc.language.iso | en | |
| dc.subject | RasGAP | zh_TW |
| dc.subject | E-Ras | zh_TW |
| dc.subject | caveolin-1 | zh_TW |
| dc.subject | caveolin-2 | zh_TW |
| dc.subject | Triton X-114分萃 | zh_TW |
| dc.subject | 蔗糖梯度離心 | zh_TW |
| dc.subject | sucrose gradient ultracentrifugation | en |
| dc.subject | caveolin-2 | en |
| dc.subject | Triton X-114 separation | en |
| dc.subject | E-Ras | en |
| dc.subject | caveolin-1 | en |
| dc.subject | RasGAP | en |
| dc.title | RasGAP與Caveolins在E-ras轉染細胞中扮演的功能 | zh_TW |
| dc.title | Role of RasGAP and Caveolins in Cells Transfected with E-ras | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 95-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 柯逢春,黃娟娟,李心予,黃偉邦 | |
| dc.subject.keyword | E-Ras,RasGAP,caveolin-1,caveolin-2,Triton X-114分萃,蔗糖梯度離心, | zh_TW |
| dc.subject.keyword | E-Ras,RasGAP,caveolin-1,caveolin-2,Triton X-114 separation,sucrose gradient ultracentrifugation, | en |
| dc.relation.page | 36 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2007-07-03 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 動物學研究研究所 | zh_TW |
| 顯示於系所單位： | 動物學研究所 | |
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