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研究生:李淑琳
研究生(外文):Shu-Lin Lee
論文名稱:以分子分析方法探討胸苷酸合成酶活性部位的變異對其酵素活性的影響
論文名稱(外文):Molecular Analysis of the Alterations of Active-site Residues in Thymidylate Synthase and their Relations to Enzyme Activity
指導教授:李益謙李益謙引用關係
指導教授(外文):Eric I-chian Li
學位類別:碩士
校院名稱:國立成功大學
系所名稱:藥理學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:84
中文關鍵詞:酵素活性胸苷酸合成�@
外文關鍵詞:Thymidylate SynthaseEnzyme Activity
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胸苷酸合成酶(thymidylate synthase, TS)經由還原性甲基化轉移一個亞甲基 (methylene group),將dUMP轉變成dTMP,而dTMP為DNA的合成(synthesis)和修補 (repair)所必須。TS在細胞週期的合成期 (S phase)大量上升,此現象在快速增殖細胞中尤為明顯。因此,TS被認為是一個癌症化療的重要標的。癌症化療藥物pyrimidine analogues如:5-FU和5-FdUR,其活性代謝物FdUMP,會與TS及其輔因子CH2-THF形成一安定的化合物,抑制體內dTMP的產生,從而抑制DNA的合成。因為pyrimidine analogues被代謝後會影響TS的活性,所以被廣泛用來癌症的治療。本實驗室先前對TS基因研究中,利用V79易於被誘導產生突變的特性,以FAT培養液篩選出多種不具TS活性之突變株,其中以T163為易突變點(由threonine轉變成alanine)。TS2在受質結合位 (substrate binding site, dUMP site)發生改變,TS15和TS21在輔因子結合位 (cofactor binding site, folate site)發生改變,其TS活性明顯降低,顯示受質結合位及輔因子結合位與其TS活性具重要相關性。我利用實驗室既有的方法對突變株進行TS回復體實驗,自突變株TS2 R208K和TS12 G239D分別篩選出回復體之群聚(colony),基因序列比對結果,發現可將原本產生突變之位置進行回復,得知氨基酸208為易突變點,且所在之受質結合位為重要之TS活性決定部位,氨基酸239為易突變點,推測可能為重要的TS活性決定位置。針對folinic acid對TS活性之探討,改變FAT培養液中folinic acid濃度,可篩選出具部分TS活性之突變株,其TS的兩個主要結合位發生突變時,確實對TS活性造成影響。以V79細胞經化學致突變劑EMS處理後,篩選對5-FdUR產生抗性之突變株,其TS活性有過度表現 (over expression)的情況,得知氨基酸116,24,89和163的突變,所造成之5-FdUR抗性,而增加其TS活性。利用這些研究幫助我們瞭解哺乳類TS與其受質和輔因子間之相互作用,並增加TS對抗癌藥物5-FU產生耐藥性有更進一步的瞭解。
Thymidylate synthase (TS, EC 2.1.1.45) catalyzes the do novo biosynthesis of thymidylate, which is necessary for DNA synthesis and repair. Fluoropyrimidines, such as 5-fluorouracil (5-FU), are used extensively in cancer therapy. Drug-resistant variants of thymidylate synthase can potentially be used in gene therapy to decrease the myelosuppressive side effects of TS-directed anticancer agents. Chinese hamster V79 cells can be easily induced to become TS deficient mutants due probably to its heterozygosity. Knowledge of the catalytic mechanism and structure of TS has increased substantially over recent years. Our lab has also previously partially cloned and sequenced the Chinese hamster TS gene, in the present study I want to use random mutagenesis to gain structure-function information about the TS molecule. I treated Chinese hamster V79 cells with EMS and isolated TS mutants with a special selection FAT medium (folinic acid, aminopterin, thymidine).The TS mutants─TS2, TS15 and TS21─have low TS activities (TS-) and contain mutations within substrate binding site and cofactor binding site. Then I treated these TS deficient mutants with EMS and selected with a thymidine deficient medium, some TS revertant colonies were selected, the TS activities of these TS revertants are interestingly reversed. I also tried to select the TS mutants with elevated folinic acid in the FAT medium and found that these FAT resistant clones have partial TS activities. Finally I mutagenized V79 cells and treated them with 5-FdUR to obtain drug-resistant mutants. The TS activities of these drug-resistant mutants are the same or a bit higher than the wild-type V79 cells. These results illustrate the intricate relations between TS enzyme activity and mutations in the substrate and cofactor binding sites as well as mutations in these sites and the 5-FdUR resistance.
第一章 序論……………………………………………………1

第二章 研究目的與策略………………………………………7

第三章 實驗材料………………………………………………8

第四章 實驗方法………………………………………………11

第五章 實驗結果………………………………………………32

第六章 討論……………………………………………………39

第七章 參考文獻………………………………………………44
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黃柏燈著,中國田鼠胸�P酸合成�@基因的選殖,定序與表現,國立成功大學藥理學研究所,碩士論文 (1997)。
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