臺灣博碩士論文加值系統

中文摘要
Ras蛋白的N端鳥糞嘌呤核甘酸結合區發生點突變時，可因發生位置的不同、取代的胺基酸不同，造成GTP-locked程度、內生性GTPase活性及細胞轉型效率(transforming efficiency)的不同，又因Ras蛋白能調控細胞的生長，因此我對具高經濟效益的養殖作物：斑節蝦，進行ras cDNA的選殖，並進一步瞭解其蛋白產物的特性及其反應條件，以利探討"斑節蝦Ras蛋白N端鳥糞嘌呤核甘酸結合區與其C端被牻牛兒牻牛兒基化的作用關係"。
首先，由Genetics Computer Group DataBank查出並比對脊椎動物H-, N-, K-Ras蛋白的胺基酸序列，從高相似性區(homologous region)設計一組鹼基輪替性引子組(degenerate primers set)，並以Polymerase Chain Reaction從斑節蝦肝胰臟得到一275 bp的ras cDNA片段，以此片段為探針由斑節蝦肝胰臟基因庫中篩得ras cDNA：全長1,294 bp, 含碼區為564 bp，經轉譯為187個胺基酸組成，與同屬無脊椎節肢動物的豐年蝦(Artemia) Ras、果蠅(Drosophila) Ras1及哺乳動物(Human、Mouse、Rat)的KB-Ras間的相同程度(identity)很高，可達79 %以上。 此斑節蝦Ras蛋白(S-Ras)的C端最後四個胺基酸: CIVF序列，為牻牛兒牻牛兒基轉移■I (Protein Geranylgeranyltransferase I)的作用位置，又其C端在165~187 a.a.間為帶有4個離胺酸和6個精胺酸的正電胺基酸散布區，與KB-Ras在165~188 a.a.間的聚離胺酸區(poly-lysines region: 含12個lysine, 有6個相鄰)不同，此區推測是與Ras蛋白穩定結合在特定的細胞膜組成有關。
再者，為了進一步瞭解S-Ras蛋白的基本特性及其反應條件，利用細菌表現S-Ras融合蛋白(N端有4 kDa的Calmodulin-binding peptide-tag, 以利純化)，經HPLC分析得知此S-Ras融合蛋白是未結合有GTP或GDP的Ras蛋白。 其GTP、GDP結合的最適情形是在pH 8.5時，GTP可達75 %、GDP達12.5 %的飽和結合率，又由解離常數值(K2：dissociation rate constant)：S-Ras-GTP (K2=1.46 x 10-3S-1) < S-Ras-GDP (K2=5.42 x 10-3S-1)，顯示S-Ras融合蛋白較喜歡結合GTP；而測試結合條件中Mg2+抑制S-Ras融合蛋白結合GTP與二價金屬離子Mn2+、Mg2+強烈抑制GTP被結合的結果相符，但是和哺乳動物(Human、Mouse、Rat) Ras蛋白的結果不同。 在intrinsic GTPase活性表現的最佳狀況與二價金屬離子的影響測試，發現在pH 6.0和適量Mg2+(0.5~1mM)是有助GTPase活性的表現，且Mn2+助力更大可達Mg2+的2.5倍。
接著，針對斑節蝦Ras蛋白N端鳥糞嘌呤核甘酸結合區設計了G12V、Q61K、N116I三個點突變蛋白。 相對於哺乳動物(Human、Mouse、Rat) Ras蛋白在同樣的位置發生該點突變時，皆能引起NIH/3T3細胞的轉型及形成transformed foci，依Ras蛋白立體結構與GTP或GDP結合關係得知：G12是位在與α-,β-phosphate作用相關的G1 Box (GXXXXGKS/T)，Q61位於G3 Box (DXXG)旁與GTP的γ-phosphate作用及Mg2+結合有關，N116位在G4 Box (NKXD)和guanine ring的辨識相關，而G12V與Q61K致使intrinsic GTPase活性降低成為GTP-locked、N116I影響GDP、GTP的結合穩定性，推測是引起細胞轉型的原因。 以Thin layer chromatography分析S-Ras和mutants融合蛋白的GTP-locked程度時，發現Q61K>G12V>S-Ras的GTP-locked能力和N116I不結合GTP、GDP的特性。 將斑節蝦肝胰臟部分純化所得的牻牛兒牻牛兒基轉移■ I，對S-Ras和mutants融合蛋白進行作用，發現GTP-locked程度愈強則被牻牛兒牻牛兒基化的程度愈高，因此我除了發現斑節蝦Ras蛋白需先結合鳥糞嘌呤核甘酸，再被牻牛兒牻牛兒基化的作用順序外，更證實了"斑節蝦Ras蛋白N端鳥糞嘌呤核甘酸結合區發生點突變時，會影響其C端被牻牛兒牻牛兒基化作用的情形"。
最後，把S-ras和mutants各別轉染至BALB/3T3細胞，並進行致使細胞轉型形成transformed foci的實驗，結果證明斑節蝦ras確有其細胞生理活性，因為S-Ras(Q61K)有使細胞轉型形成transformed foci的能力，又發現此transformed foci細胞中，參與glycolysis的Glyceraldehyde-3-phosphate dehydrogenase mRNA表現量比對照組未轉型細胞高過50 %，推測是與cancer cell 的proliferation有關。 而S-Ras(G12V)和S-Ras(N116I)的結果則與文獻中哺乳動物(Human、Mouse、Rat)點突變ras會使細胞轉型形成transformed foci的結果不同。 對於S-Ras(Q61K)可使細胞轉型形成transformed foci的推測是，Q61K突變點的位置有助於與細胞轉型的訊息傳遞相關蛋白結合所致，至於此關鍵蛋白為何﹖ 則是下一個要探討的目標。 另外，我試圖以BALA/3T3 transformed foci實驗的結果來解釋如下的假設：S-Ras點突變蛋白的GTP-locked程度愈強、被牻牛兒牻牛兒基化的程度愈高時，是否會造成細胞轉型形成transformed foci的能力就愈大﹖ 結果發現：假設在此無法獲得證實，原因是斑節蝦Ras蛋白C端序列有其特異性，所以用蝦子的細胞株做此實驗較為正確，而這也正是想要建立蝦子細胞株的原因。

Abstract
The identity and the specific location of the point mutations in the guanidine nucleotide-binding region of Ras protein N-termini affected the Ras GTP-locked ability, GTPase activity, and moreover the efficiency of cellular transformation. In order for the Ras protein to promote cell proliferation it has to be recruited to the cell membrane for activation. Prenylation of the Ras C terminus is a pivotal event, since it introduces a hydrophobic tail for insertion into the cell membrane. Therefore, I attempted to study the effect of point mutations in the guanidine nucleotide binding region on the C-terminal geranylgeranylation of Penaeus (Marsupenaeus) japonicus Ras.
First, I designed the degenerate primer set base on the homologous region of the vertebrate H-, K-, and N-Ras amino acid sequences from the Genetics Computer Group Data Bank. I obtained a 275 bp PCR product from the hepatopancreas cDNA of the banded prawn Penaeus (Marsupenaeus) japonicus. I used this PCR product as a probe to screen the cDNA library from the hepatopancreas of the banded prawn Penaeus (Marsupenaeus) japonicus and obtained a ras cDNA with a full length of 1,294 bp and a 564 bp open reading frame. Translation of the coding region gave a to 187-residue shrimp Ras protein(S-Ras). S-Ras shared at least 79 % identity with Artemia Ras, Drosophila Ras1 and mammalian(human, mouse, rat) KB-Ras. The C-terminus of the S-Ras contains two signal motifs: one motif (CIVF box) comprising the last four amino acids of the C-terminus was the protein geranylgeranyltransferase I recognition and reaction site. The other motif contains four lysines and six arginines that is located at the 165~187 residues of the C-terminus, and this motif was different from the poly-lysine region of the C-terminus of the mammalian(human, mouse, rat) KB-Ras protein, implying that the polybasic amino acid region of the Ras proteins interacted with species specific membrane anchorage domains or sites.
I expressed the S-ras gene in E.coli with a 4 kDa calmodulin-binding peptide tag at the N-terminus. The p25 S-Ras fusion protein was purified with the calmodulin affinity column. The optimal GTP/GDP binding condition was found to be at pH 8.5; the saturated binding rate of GTP was 75 % and that of GDP was 12.5 %. The dissociation rate constant of the S-Ras-GTP complex (K2=1.46 x 10-3S-1) was smaller than the S-Ras-GDP complex (K2=5.42 x 10-3S-1), which means that the S-Ras fusion protein prefers to bind to GTP over GDP. The GTP binding activity of the S-Ras fusion protein was strongly inhibited by Mg2+ or Mn2+, in contrast with the previous studies done with the mammalian(human, mouse, rat) Ras proteins. The optimal pH for GTPase activity was pH6.0; under this condition, adding 0.5~1 mM Mg2+enhanced the GTPase activity, and the activity was increased 2.5 folds when Mg2+ was replaced with Mn2+.
I constructed several mutants of the S-ras with single nucleotide point mutation: G12V, Q61K and N116I. The same mutations in the mammalian(human, mouse, rat) Ras proteins had the ability to transform the NIH/3T3 cells. The structure of Ras protein bound with GTP or GDP has been analyzed by x-ray crystallography(Sprang,1997), and it has been shown that G12 was located at the G1 Box (GXXXXGKS/T) and associated with α-,β-phosphate, that Q61 was nearby the G3 Box (DXXG) and associated with γ-phosphate and Mg2+, and that N116 was located at the G4 Box (NKXD) and recognized the guanine ring. The G12V and Q61K mutants have a reduced intrinsic GTPase activity, and thus became GTP-locked, active form of Ras. The GDP/GTP exchange rate of N116I was increased. Therefore, the mutants of Ras were constitutively activated and passed down the growth signal in cells. I detected the GTP-locked ability of the wildtype and mutant Ras fusion proteins with thin layer chromatography. The results showed that the GTP binding activities were Q61K > G12V > wildtype and that N116I did not have the GTP/GDP binding activity. The protein geranylgeranyltransferase I(PGGT I) partially purified from the hepatopancreas of the shrimp Penaeus (Marsupenaeus) japonicus was able to react with the wildtype and mutant S-Ras fusion proteins, the extent of geranylgeranylation positively correlates with the GTP-locked ability. It was thus proven that the point mutations in the guanidine nucleotide-binding region of the S-Ras protein N-termini affected the C-terminal geranylgeranylation efficiency.
Finally, the wildtype and mutant S-ras genes were transfected to the BALB/3T3 cells and tested for transformed foci formation. The S-ras(Q61K) had the ability to transform the BALB/3T3 cells and this result shows the S-ras had biological activity. The other two mutants: the S-ras(G12V) and the S-ras(N116I) did not form transformed foci, which differs from the mammalian(human, mouse, rat) Ras mutants, which with the same mutant sites and could transform the NIH/3T3 cells. When I analyzed the S-ras(Q61K) transformed foci cells, I found the mRNA level of the Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to be 50% higher than non-transformed controlled cells. This enzyme is involved in the glycolysis pathways and is related with the proliferation of cancer cells. Although data from the BALB/3T3 cells did not provide convincing evidence for my hypothesis, that increasing the GTP-locked ability of the S-Ras proteins could enhance their C-termini geranylgeranylation, and thus enhance the transforming efficiency, it should be noted here that using the mammalian cells to study shrimp Ras cellular function may be unsuitable. Since the C-termini of the S-Ras and the mammalian(human, mouse, rat) Ras are very different, they may prefer to bind specific components of their own cell types, for example, cell membrane or docking proteins. The postion and identity of the mutation I introduced may affect S-Ras''s affinity for its regulatory proteins and/or down-stream effectors in addition to GTP-locked ability. This may explain the lack of correlation between GTP-locked ability and transformation efficiency of the different mutants I studied. We would therefore like to establish a shrimp cell line for further studies.

封面
目錄
中文摘要
英文摘要
緒論
Ras蛋白的基本特性與一級結構分布
Ras蛋白結晶的立體結構
Ras蛋白N端的結構特性及重要性
Ras蛋白C端的結構特性及重要性
異戊烯基轉移？的種類及受質蛋白的特徵
蛋白分子內N端與C端的結構關聯性
實驗目的
器材與藥品
一.材料來源
二.器材與儀器
1.器材
2.儀器
3.資料庫
三.藥品來源
1.合成胜？
2.藥品與試劑
3.Primer合成與菌株
方法
總RNA的抽取
訊息RNA的分離
首股互補DNA的合成
DNA聚合？連鎖反應增生DNA片段
蝦子ras cDNA探針的獲得
1.PCR產物接於pMOS Blue T-vector
2.質體轉型至MOS Blue勝任細菌
3.質體的小量產及分離
4.質體內PCR產物的定序
5.合成Dig-11-dUTP標定的蝦子ras cDNA探針
南方點墨分析法
甲醛膠電泳分析RNA
北方點墨分析法
蝦子肝胰臟cDNA基因庫的建構
由基因庫篩取蝦子ras cDNA
蝦子ras cDNA的3''端快速量化及序列測定
Mammalian Ras cDNA的獲得及表
斑節蝦ras cDNA的細菌表現質體構築
斑節蝦Ras融合蛋白的表現
細菌包涵體內融合蛋白的分離與純化
蛋白質定量
Tricine-硫酸十二酯鈉聚丙烯醯氨膠電泳分析和自動放射分析圖
Coomassie Blue R-250染色法
西方點墨分析法
斑節蝦Ras融合蛋白的定性分析
1.HPLC分析E.coli表現的斑節蝦Ras融合蛋白中GNP結合的情形
2.斑節蝦Ras融合蛋白結合鳥糞嘌呤核甘酸的最適狀況調查
3.二價金屬離子對斑節蝦Ras融合蛋白與GNP結合的影響
4.斑節蝦Ras融合蛋白內生性GTPase活性的最適表現調查
5.二價金屬離子對斑節蝦Ras融合蛋白的GTPase活性表現的影響
斑節蝦單鹼基點突變ras cDNA的細菌表現質體建構
DNA定序法(SequenaseTM Version 2.0 Kit;USB)
斑節蝦單鹼基點突變ras cDNA的限制？鑑定
斑節蝦Ras融合蛋白的GTP-locked程度測試
斑節蝦肝胰臟的蛋白質牻牛兒牻牛兒基轉移？I部分純化與西方點墨法檢測
斑節蝦蛋白質牻牛兒牻牛兒基轉移？I活性檢測
斑節蝦Ras融合蛋白被牻牛兒牻牛兒基化作用
細胞培養
斑節蝦ras及其單鹼基點突變cDNA的細胞表現質體建構
斑節蝦ras及其mutants的BALB/3T3細胞轉染
轉型細胞團形成測試(Transformed foci assay)
篩選轉染有斑節蝦ras或其mutants的BALB/3T3細胞株群
轉染細胞株群中斑節蝦ras(Q61K)mutant mRNA的北方點墨法檢測
轉染細胞株群中斑節蝦ras(Q61K)mutant蛋白的免疫沈澱法及西方點墨法檢測
結果
一.斑節蝦ras cDNA的選殖
1. 蝦子ras cDNA片段探針的獲得
2.斑節蝦ras cDNA的選殖
二.斑節蝦Ras融合蛋白的表現及定性測試
1.斑節蝦ras cDNA含碼區接進pCAL-n-EK表現載體
2.斑節蝦ras cDNA於E.coli的表現及純化與分析
3.HPLC分析E.coli表現的斑節蝦Ras融合蛋白中結合GNP的情形
4.斑節蝦Ras融合蛋白的GTP/GDP結合能力測試
5.斑節蝦Ras融合蛋白的GTPase活性測試
三.斑節蝦Ras蛋白N端鳥糞嘌呤核甘酸結合區與其C端被牻牛兒牻牛基化的探討
1.斑節蝦單鹼基點突變ras cDNA的設計及蛋白的合成
2.斑節蝦Ras融合蛋白與其點突變融合蛋白被牻牛兒牻牛兒基化作用
四.斑節蝦ras及其單鹼基點突變cDNA在BALB/3T3細胞的表現
1.BALB/3T3細胞的轉型細胞團生成測試
2.檢測轉染有斑節蝦ras(Q61K)mutant的BALB/3T3細胞中ras的表現
討論
一.斑節蝦ras cDNA的選殖
1.斑節蝦Ras蛋白C端序列特性與其融合蛋白的電泳膠分析
2.斑節蝦ras cDNA專一性探針對其組織器官做北方點墨分析
二.斑節蝦Ras融合蛋白的表現及定性測試
1.斑節蝦Ras融合蛋白的GNP結合特性
2.斑節蝦Ras融合蛋白的GTPase活性測試
三.斑節蝦Ras蛋白N端鳥糞嘌呤核甘酸結合區與其C端被牻牛兒牻牛基化的探討
1.斑節蝦點突變Ras融合蛋白的GTP-locked特性測試
2.斑節蝦Ras融合蛋白與其點突變融合蛋白被牻牛兒牻牛兒基化作用
四.斑節蝦ras及其單鹼基點突變cDNA在BALB/3T3細胞的表現
1.BALB/3T3轉型細胞團生成測試
2.檢測斑節蝦ras(Q61K)mutant在BALB/3T3細胞的表現
總結
展望
參考文獻
表
圖

參考文獻
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