臺灣博碩士論文加值系統

核糖蛋白L5 (ribosomal protein L5)大小約為34kDa，是組成真核細胞核糖體60S次單位的一員，L5主要負責攜帶5S ribosomal RNA (5S rRNA) 及其在細胞核內外的運送。L5除了具有運送5S rRNA的功能外，亦被指出能和細胞內的一些蛋白交互作用，如:casein kinase Ⅱ、typeΙphosphatase及mdm2-p53，過去的研究報告指出RPA2的磷酸化會造成原本結合在RPA1上的p53被釋放出來，又L5被發現會與mdm2和mdm2-p53複合體交互作用，且學長發現當UV照射細胞時，磷酸化的replication protein A2(RPA2)會與L5形成一個複合體，於是我們推測在一般狀況下，L5可能與p53結合，當UV照射細胞後，L5離開p53，使得p53因此活化，而進行p53-dependent checkpoint control。因此我們利用TNT Quick Coupled Transcription/Translation Systems與免疫沉澱法(immunoprecipitation)來偵測L5與p53蛋白是否有直接的交互作用，然而從實驗結果中我們並沒有看到L5與p53有直接地交互作用。且為了看經UV照射後L5與p53結合的關係，我們以50J/m2 的UV 照射細胞，經不同時間點收細胞以便進行西方墨點法及免疫沉澱法來看其p53、mdm2及L5的變化，進而了解它們的關係。由其結果顯示，當UV照射細胞後2小時，p53就會有明顯的增加，而mdm2卻要在4小時後才會消失，於是我們認為當UV照射細胞後2小時p53的增加，有一部分除了來自於原本與mdm2結合的p53被釋放出來的以外，另一部分可能是從與另一個蛋白結合中被釋放出來的p53。
創傷弧菌(Vibrio vulnificus)CKM1菌株的藍色螢光基因(bfpvv)其轉譯的藍色螢光蛋白(BFPVv)可經由長波UV燈照射而發出顯著的藍色螢光，且在bfpvv 5’端上游發現其調節基因，bfpvvR，此調節蛋白屬Lys family調節蛋白的一員。純化的BFPVvR蛋白可與bfpvv 5’端上游區域(即bfpvvR 3’端下游區域)產生結合，且我們發現BFPVv蛋白的表現量在bfpvvR mutant URD101內比wild type CKM-1還高，這意謂著BFPVvR可能是扮演著一調節者的角色來調控bfpvv 基因的表現。為了進一步探討BFPVvR蛋白如何調控BFPVv，於是我們利用primer extension analysis來找出其transcriptional start site。由結果得知bfpvv mRNA在轉譯起始點上游88個氮鹼基的位置開始轉錄。且為了進一歩確認BFPVvR是否扮演著一負調節者的角色來調控bfpvv gene的表現，於是我們將含有bfpvv的promoter 融合GST並構築在pBR322質體(PG/ pBR322)，然後觀察GST在CKM-1(PG/ pBR322)及URD101(PG/ pBR322)內的表現，由結果發現GST蛋白在URD101的表現量比在CKM-1來得高，於是我們初步推測BFPVvR是扮演著一負調節者的角色來調控bfpvv gene的表現。

Ribosomal protein L5 (L5) is a 34KDa large subunit protein of 60S ribosome. L5 is known to bind specifically to 5S rRNA and is involved in nucleocytoplasmic transport of this rRNA. In addition to its 5S rRNA transport activity, L5 has also been shown to bind to other cellular proteins that participate in various intracellular transport pathways. For example, L5 interacts with the β subunit of casein kinase Ⅱ (CKⅡβ), typeⅠprotein phosphatase and mdm2-p53. Previous studies indicated that phosphorylation of replication protein A2 (RPA2) prevented the association of RPA with p53, and L5 was associated with mdm2 and mdm2-p53 complexes. In previous studies, we discovered that L5 interacted with hyperphosphorylated form but not hypophorylated form of RPA2 when cells were irradiated with UV. This raises an interesting possibility that L5 may be directly associated with p53 in unirradiated cells, and upon UV irradiation, L5 releases the bound p53, thus transducing the damage signal and activating the p53-dependent checkpoint control. In this study, TNT® Quick Coupled Transcription / Translation Systems and immunoprecipitation were performed to investigate whether L5 directly interact with p53 or not. The results of our primary data indicated that L5 associate with, but does not directly interact with p53. To test the effect of UV radiation on L5 associate with p53, cell lysates were prepared at different times after UV irradiation at 50J/m2. Western blot analysis and immunoprecipitation were performed. The results showed that p53 accumulated at 2 hr, but mdm2 disappeared at 4 hr after UV radiation. Thus, we thought that about half of p53 accumulation may be not released from p53-mdm2 complex when UV radiation. It may be released from the other protein.
In our preliminary studies, the gene, bfpvv, encoding a blue fluorescent protein (BFPVv) has been cloned from Vibrio vulnificus CKM-1. E. coli XL1B. containing bfpvv exhibited blue fluorescence phenotype when excited by long-wave UV light. Sequence analysis of the upstream region of the bfpvv gene revealed a gene, bfpvvR, is transcribed divergently from the bfpvv and its putative amino acid sequence was similar to the LysR family of transcriptional regulators. A DNA mobility shift assay showed the binding of purified BFPVvR to the bfpvvR-bfpvv intergenic promoter region. We also found that the expression of BFPVv in the bfpvvR mutant URD101 significantly increased compared to wild type CKM-1, indicating that BFPVvR may be a repressor for the bfpvv gene. To understand how BFPVvR regulates BFPVv, in this study, a primer extension analysis was performed to determine transcriptional start site. The results showed that the transcriptional start site of bfpvv mRNA was at the position -88 relative to the translational start site. To confirm that BFPVvR is a repressor for the expression of the bfpvv, a Pbfpvv-gst fusion was constructed on plasmid PG/pBR322, and the expression of gst in wild type strain containing PG/pBR322 and in URD101 containing PG/pBR322 were examined. The results revealed that the expression of GST in the URD101(PG/pBR322) significantly increased compared to those in wild type CKM-1(PG/pBR322), indicating that BFPVvR is a repressor for the expression of the bfpvv.

授權書
口試合格證明
中文摘要 Ⅰ
英文摘要 Ⅲ
致謝 Ⅴ
目錄 Ⅵ
圖表目錄 Ⅸ
縮寫檢索表 Ⅹ
第一章 選殖與L5交互作用之蛋白 1
緒論 2
材料與方法 4
一、使用之菌株、載體及培養基 4
二、質體DNA在大腸桿菌的轉形作用 7
三、少量質體DNA的抽取 8
四、基因庫質體DNA的抽取 10
五、GAL4-BD-L5 融合蛋白的構築 11
六、限制酶切割質體DNA 12
七、接合反應 13
八、酵母菌的共同轉形作用 13
九、-galactosidase filter assay 15
十、酵母菌質體DNA的抽取 16
十一、篩選含GAL4活化區融合蛋白的質體DNA 17
結果 18
討論 20
第二章 L5與P53之交互作用 22
緒論 23
材料與方法 25
一、L5、CKⅡ及p53全長和截短片段重組蛋白的構築 25
二、試管內( in vitro )表現放射線標定的重組蛋白 28
三、免疫沉澱法 29
A、利用TNT System表現出來的蛋白質做免疫沈澱法 30
B、經UV damage後收細胞做免疫沈澱法 31
四、細胞培養 31
A、Subculture(以10cm培養皿為例) 32
B、凍細胞 32
C、解凍細胞 33
D、UV照射細胞(以10cm培養皿為例) 33
E、收細胞(Harvesting cell lysate) 33
五、蛋白質濃度定量 34
六、蛋白質電泳製作 34
七、西方墨點法 36
結果 40
一、利用免疫沈澱法來偵測 L5與不同片段p53的交互作用 40
二、利用免疫沈澱法來偵測不同片段 L5與p53的交互作用 40
三、利用免疫沈澱法來偵測L5與GSTp53c的交互作用 41
四、UV damage後p53與mdm2的變化 41
討論 43
一、利用免疫沈澱法來偵測 L5與p53交互作用的情形 43
二、L5與mdm2、p53的關係 45
第三章 螢光蛋白與其調節因子之研究 46
緒論 47
材料與方法 50
一、使用的菌株、載體及培養基 50
二、質體DNA的抽取 51
三、菌體RNA的抽取 52
四、甲醛洋菜膠體電泳 54
五、核酸引子延伸實驗 56
A、Primer labeling 57
B、DNA marker labeling 57
C、Primer extension reaction 58
六、DNA定序 58
A、DNA定序 58
B、製作定序膠體 60
C、DNA定序的電泳分析 61
七、將bfpvv promoter融合GST並構築在pBR322載體上 61
八、電穿孔法 63
結果 65
一、創傷弧菌CKM-1與URD101產生藍色螢光情形 65
二、 以電穿孔法將promoter-GST/pBR322送入CKM-1與URD101並觀察其GST表現情形 65
三、創傷弧菌藍色螢光基因轉錄起始點的決定 66
討論 67
參考文獻 69
圖表 74
自述 88

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