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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃火鍊(Fore-Lien Huang) | |
| dc.contributor.author | Chia-Hsiung Cheng | en |
| dc.contributor.author | 鄭嘉雄 | zh_TW |
| dc.date.accessioned | 2021-06-15T04:06:11Z | - |
| dc.date.available | 2010-02-24 | |
| dc.date.copyright | 2010-02-24 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-02-08 | |
| dc.identifier.citation | References
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45143 | - |
| dc.description.abstract | 在本論文中，我們選殖出豐年蝦的蛋白激酶JAK與轉錄因子STAT，經由胺基酸的序列比對，豐年蝦與果蠅的蛋白激酶，雖然只有19%的相同性及33%的相似性，但是包含有完整的七個蛋白激酶保留性區域(JAK homology domain, JH domain)。另外，豐年蝦與果蠅的轉錄因子，則有較高的30%相同性。由這些低相同性的結果推測，無脊椎動物中不同的物種，在其蛋白激酶與轉錄因子具有其特異性。RT-PCR分析的結果顯示，豐年蝦蛋白激酶與轉錄因子在發育過程中呈現持續性的表現，與果蠅的蛋白激酶與轉錄因子在發育過程中表現特性相同。另外，我們也將豐年蝦蛋白激酶的JH1區域接合到轉錄因子的碳端(C-terminal)末端，構築了持續性活化的轉錄因子，AfSTAT-JH1, 可自行活化其酪胺酸處(tyrosine residue)，進而具有結合到果蠅Raf啟動子上的轉錄因子特定結合區域的能力。在SF9昆蟲細胞中，包含豐年蝦蛋白激酶和轉錄因子都具有轉錄活化(transactivation)果蠅Raf和totA啟動子的能力。然而，酪胺酸處磷酸化的活化態豐年蝦轉錄因子並未偵測到，經由細胞原位分析實驗顯示，大部份的豐年蝦轉錄因子都停留在細胞質中。我們的結果證實，包含蛋白激酶和轉錄因子都存在於豐年蝦的基因體中，將來對於研究JAK/STAT訊號傳遞路徑在豐年蝦的發育與免疫反應，提供了一個重要的基礎。 | zh_TW |
| dc.description.abstract | In this study, we isolated and characterized both JAK and STAT genes from Artemia, Artemia franciscana. Although AfJAK showed only 19% identity (33% similarity) to the Drosophila Hop protein, AfJAK contained the characteristic JAK homology domain (JH domain) from JH1 to JH7. On the other hand, AfSTAT showed higher identity (30%) to Drosophila STAT (STAT92E). The low identities of AfJAK and SfSTAT to Drosophila Hop and STAT92E suggest that JAK and STAT proteins are unique in each different species of invertebrate. RT-PCR analysis showed that both AfJAK and AfSTAT transcripts were ubiquitously expressed in the embryo, which is similar to the expression patterns of Drosophila Hop and STAT92E mRNAs during development. In addition, we generated a constitutively active form of AfSTAT by fusing the JH1 domain of AfJAK to the C-terminal end of AfSTAT. This fusion protein, AfSTAT-JH1, autophosphorylated on its tyrosine residue and was able to bind to specific DNA motifs including the STAT-binding motifs in the Drosophila Raf promoter. Both AfJAK and AfSTAT proteins elicited the transactivation potential toward the fly Raf and totA promoter in Sf9 cells. However, tyrosine phosphorylation of AfSTAT was not detected, which is consistent with the cellular localization analysis that most AfSTAT proteins were in the cytoplasm. Our results demonstrate that both JAK and STAT are present in the genome of Artemia, which can serve as the basis for further investigations to explore the role of the JAK/STAT signal pathway in the development and immune response of brine shrimp. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T04:06:11Z (GMT). No. of bitstreams: 1 ntu-99-D93b43003-1.pdf: 4606597 bytes, checksum: 605e130be4d902b1f11a6d2916e3d5d3 (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | 中文摘要 1
Abstract 2 Abbreviation 4 Introduction 5 The importance of Artemia 5 The Life Cycle of Artemia 6 The immune responses in Artemia 6 The JAK/STAT signal pathway in mammals 7 The JAK protein family 8 The STAT protein family 8 Specific aims 16 Materials and Methods 17 Total RNA isolation and first-strand complementary (c)DNA synthesis 17 Cell culture 17 Preparation of nuclear extracts 18 Isolation of the JAK and STAT cDNA fragments of A. franciscana 18 Plasmid construction 19 Rapid amplification of cDNA ends (RACE) 20 RT-PCR 21 Sequence analysis and phylogenetic tree analysis 22 Isolation Drosophila totA promoter from genomic DNA 22 Expressions of AfJAK-HA, AfSTAT-HA, and AfSTAT-HA-JH1 in cultured cells 23 Western blot analysis 23 Subcellular localization and image analysis 24 Transactivation assay 24 Electrophoretic mobility shift assay (EMSA) 25 Results 26 Cloning and characterization of the AfJAK and AfSTAT genes 26 Phylogenetic analysis of the AfSTAT and AfJAK proteins 27 Expression profiles of the AfJAK and AfSTAT transcripts at different developmental stages 28 Expression and DNA-binding property of the AfSTAT-HA-JH1 fusion protein that is constitutively active in tyrosine phosphorylation 29 Both AfJAK and AfSTAT proteins displayed similar transactivation abilities toward the Drosophila Raf promoter in Sf9 cells 31 Both AfSTAT-HA and AfSTAT-HA-JH1 proteins displayed similar transactivation abilities toward the Drosophila totA promoter in Sf9 cells 33 Conclusion and perspective 41 Figures 43 Figure 1. Alignment of deduced amino acid sequences of AfJAK with those of human JAK family members and Drosophila hopscotch (Hop). 44 Figure 2. Alignment of deduced amino acid sequences of AfSTAT with those of STAT proteins from different species. 46 Figure 3. Phylogenetic tree analysis of JAK family proteins from invertebrate and vertebrate species. 48 Figure 4. Phylogenetic tree analysis of STAT proteins from various species. 50 Figure 5. Expression profiles of AfJAK and AfSTAT transcripts in embryos by RT-PCR. 51 Figure 6. Western blot analysis of total cell lysates from cells transfected with pCMV-AfSTAT-HA, pCMV-AfSTAT-HA-JH1, and mock as a negative control. 52 Figure 7. Intracellular localization of AfJAK-HA, AfSTAT-HA, and AfSTAT-JH1 in COS-1 cells by fluorescent microscopy. 54 Figure 8. Electrophoretic mobility shift assay (EMSA) of the AfSTAT-HA and AfSTAT-HA-JH1 fusion protein. 56 Figure 9. Transactivation abilities of the AfJAK and AfSTAT proteins toward a fly Raf promoter in Sf9 cells. 58 Figure 10. Western blot analysis of total cell lysates from cells transfected with pIZ/V5-AfJAK-HA, pIZ/V5-AfSTAT-HA, and pIZ/V5-AfSTAT-JH1. 59 Figure 11. Intracellular localization of AfJAK-HA, AfSTAT-HA, and AfSTAT-JH1 in Sf9 cells by fluorescent microscopy. 60 Figure 12. Transactivation abilities of the AfSTAT-HA and AfSTAT-HA-JH1 proteins toward a fly TotA promoter in Sf9 cells. 62 References 63 | |
| dc.language.iso | en | |
| dc.title | 豐年蝦蛋白激酶JAK與轉錄因子STAT基因表現與特性分析 | zh_TW |
| dc.title | Expression and characterization of the JAK kinase and STAT protein from brine shrimp, Artemia franciscana | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.coadvisor | 黃銓珍(Chang-Jen Huang) | |
| dc.contributor.oralexamcommittee | 李明亭(Ming-Ting Lee),黃鵬鵬(Pung-Pung Hwang),黃聲蘋(Sheng-Ping L. Hwang) | |
| dc.subject.keyword | 豐年蝦,蛋白激酶,轉錄因子,轉錄活化,免疫反應, | zh_TW |
| dc.subject.keyword | JAK,STAT,DNA binding,Brine shrimp,Artemia franciscana, | en |
| dc.relation.page | 70 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2010-02-08 | |
| 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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