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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.author | 陳正緯 | zh_TW |
| dc.date.accessioned | 2021-07-01T08:11:19Z | - |
| dc.date.available | 2021-07-01T08:11:19Z | - |
| dc.date.issued | 1998 | |
| dc.identifier.citation | Aasland, R., Gibson, T.J., Stewart, A.F. (1995) The PHD finger: implications for chromatin-mediated transcriptional regulation. Trends Biochem. Sci. 20:56-59.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75008 | - |
| dc.description.abstract | 老鼠 Requiem 基因為 IL－3 存活依賴性鼠類骨骷細胞株 FDCP-1 缺乏生存因數 IL－3 時誘導 FDCP-l 細胞株進入細胞自戕程式所必須之蛋白，屬於 d4－family 的一員。為找尋 d4－family其他成員，利用 d4－domain 比對 Genebank ，同時經由分子選殖得到一 cDNA , R5。利用北方墨點分析法可得到 R5 mRNA 在不同組織及細胞株的表現及分佈情形。以 R5 DNA 序列比對可得到小鼠與線蟲之 R5 homologs 。以 R5 之S31B 區域（胺基酸 102－288）所製作的多源抗體可免疫共沉澱 K562 細胞內之 R5 蛋白質與 R5－d4- cDNA 表現之蛋白質。以 d4－domain 表現出來的重組蛋白質（胺基酸 289 －498 ）可與 HeLa 細胞內的數種蛋白質產生交互作用，證實 d4－ domain 為 R5 與其他蛋白質的交互作用區域。 R5具有多個 DNA－dependent protein kinase ( DNA－PK ）磷酸化位置，經 DNA - PK 激?分析法證實為其良好的 in vitro 受質。 R5 可受 DNA－PK 的磷酸化推測可能在 DNA－PK 多重調節功能上扮演重要的角色。 | zh_TW |
| dc.description.abstract | Requiem, a mouse gene activated during apoptosis induced by IL-3 deprivation of the IL-3-dependent murine myeloid cell line FDCP-1, belongs to members of d4-family with a d4-domain in its C-terminus. A novel d4-family gene, human R5 was cloned based on the homology to requiem d4-domain. Mouse and C.elegans R5 homologs were also identified by blasting Genebank database with human R5 sequence. Northern blots were performed with mRNA from various human tissues and cell lines. Polyclonal antibody against S31B region (aa.102-288) could be used for immunoprecipitating R5 from K562 cells and in vitro transcription/translation product of R5-d4+ cDNA. Recombinant protein R5-S31d4-GST (aa.289-498) containing d4- domain was found to interact with various proteins in HeLa cells. There are at least 5 putative DNA-PK phosphorylation sites in R5 sequence and were identified. The fact that R5 protein can indeed be phosphorylated by DNA-PK in vitro suggests that DNA-PK may serve as a potential regulator of R5 in vivo. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-01T08:11:19Z (GMT). No. of bitstreams: 0 Previous issue date: 1998 | en |
| dc.description.tableofcontents | 目錄. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I 縮寫表. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV 英文摘要. . . . . . . . . . . . . . . . . . . . . . . . . . . . . V 中文摘要. . . . . . . . . . . . . . . .. . . . . . . . . . . . .VI 第一章、緒論（ Introduction ) . . . . . . . . . . . . . . 1 第二章、材料與方法（Material and Methods ) . . . . . . . . 5 一、質體之抽取（ Plasmid Preparation ) . . . . . . . . . . . . 5 二、 SDS 聚丙烯醯胺凝膠電泳（SDS－PAGE ) . . . . . . . 6 三、製備勝任細胞及細胞轉型（ Competent Cell Preparation and Transformation ) . . . . . . . . . . . 7 四、 DNA 探針之放射性標記（ DNA Probe labeling by isotope ) . . . . . . . . 7 五、篩選 Uni－ZAP HeLa cDNA Library ( Screening of Uni－ZAP HeLa cDNA Library. . . . . 8 六、表現 GST 融合蛋白質（ Expression of Recombinant GST－ Fusion Protein ）. . . . . . . 9 七、 Glutathione ( GSH ）親和性管柱純化融合蛋白質（ Purification of GST－Fusion Protein by GSH Affinity Chromatography ). . . 9 八、 SDS 製備型管柱膠體純化融合蛋白質（ Purification of Recombinant Protein by SDS－Preparative Gel ) . . . . . . . . . . 10 九、抗 R5 多源抗體製備（ Preparation of anit－R5 polyclonal antibody ). . . . . 10 十、純化專一性抗 R5 多源抗體（ Purification of anti－R5 Specific Antibody）. . . . . . 11 十一、西方墨點法（ Western Blot ) . . . . . . . . . . . . . . . . . . . 11 十二、免疫沉澱法（ Immunoprecipitation ) . . . . . . . . . . . . 11 十三、多種組織、多種細胞株的北方墨點分析（ Multiple Tissues and Multiple Cell Lines Northern Blot ) . . . . . . . . . . . . . . . . . . . . . . . 12 十四、細胞培養（ Cell Culture ) . . . . . . . . . . . . . . . . . . 12 十五、製備全細胞萃取物（ P reparation of Whole Cell Extract ) . . . 13 十六、製備細胞核萃取物（P reparation of Nuclear Extract ) . . . . . . . . . . . . . 13 十七、免疫螢光細胞染色法（ Immunofluorescence Cell Staining ) . . 13 十八、 DNA 定序法（ D NA Sequencing ) . . . . . . . . .. . . . . . . . . 14 十九、離體轉錄和轉譯反應（ in vitro transcription and translation ) . .14 二十、遠西方墨點法（ Far－Western Blot ) . . . . . . . . . . . . . . . 15 二十一、離體 DNA－PK 激醃分析法（ in vitro DNA－PK kinase assay ) . . . 16 二十二、 5 ’端快速放大 cDNA 端點（ 5’－Rapid Amplification of cDNA Ends ) . . . . .16 第三章、結果（ R esult ) . . . . . . . . . . . . . . . . 17 一、d4－domain 之 DNA 序列比對. . . . . . . . . . . . . . . . . . 17 二、 hR5－d4+與 hRS－d4- 之 cDNA 選殖. . . . . . . . . . . . 17 三、老鼠與線蟲（ C . elegans ) R5 homologs 之發現. . . . . . . . . 19 四、R5 重組蛋白質之表現與純化. . . . . . . . . . . . . . . . . . . . . 19 五、hR5－d4+之 in vitro transcription and translation . . . . . . . . 20 六、抗 R5 多源抗體可免疫沉澱 K562 細胞內 R5 蛋白質. . . . . . .20 七、各組織與細胞中 hR5 mRNA 的表現情形. . . . . . . . . . . . . . . . . . 21 八、d4－domain 參與蛋白質間的交互作用. . . . . . . . . . . . . . . . . . . 21 九、R5 為 DNA－PK in vitro 受質. . . . .. . . . . . . . . . . . . . . . . . . . 22 第四章、討論（ Discussion ) . . . . . . . . . . . . . . . . . . . . . . . . 23 一、 S25 可能為全長（ full－length ）的 RS－d4+ cDNA . . . 23 二、 R5－d4 ＋與 RS－d4－之關係. . . . . . . . . . . . . . . . . . 24 三、 D4－domain 參與蛋白質間交互作用. . . . . . . . . . . . 25 四、 R5蛋白質在細胞中的表現. . . . . . . . . . . . . . . . . . . . . . . 26 五、 R5 的生化及生物功能. . . . . . . . . . . . . . . . . . . . . 26 六、 R5 未來的研究方向. . . . . . . . . . . . . . . . . . . . . . 28 參考資料. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 圖一、 d4－domain DNA 序列比對結果. . . . . . . . . . . . . . . . 36 圖二、嵌在 pBluescript SK ( + ／一）載體上的 R5－EST ( 978bP ) cDNA. . . . . . . . . 37 圖三、 hR5－d4+之 CDNA 序列與胺基酸序列. . . . . . . . . . 38 圖四、 hR5－d4+與 mouse : equiem/ubi-d4 , human neuro-d4 之 d4-domain 比較. . . . . . 40 | |
| dc.language.iso | zh-TW | |
| dc.title | 選殖及鑑定一含 d4－domain 之基因 | zh_TW |
| dc.title | C1oning and Characterization of a cDNA encoding a protein with d4－domain . | en |
| dc.date.schoolyear | 86-2 | |
| dc.description.degree | 碩士 | |
| dc.relation.page | 70 | |
| dc.rights.note | 未授權 | |
| dc.contributor.author-dept | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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