染色體的基本組成單元是由H2A、H2B、H3、H4各二分子所組成

的核心組蛋白纏繞著146 鹼基對DNA而成。核心組蛋白的共價修飾藉

由貢獻額外的遺傳訊息來調節基因組的功能。現階段，組蛋白常見

的共價修飾之一是甲基化。近年來，確定甲基化是具有可逆性的，

因此發現了另一種機動性組蛋白共價修飾，即去甲基化。所以，組

蛋白的共價修飾在基因表現的遺傳控制上扮演一個極關鍵的角色。

本研究探討啤酒酵母菌去甲基酶JHD1p的受質專一性研究，基

於報導在酵母菌的胞外去甲基酶JHD1p為具活性的酵素，而且大部份

研究都以組蛋白上的受質為主要研究目標，所以本實驗想利用啤酒

酵母菌去甲基酶JHD1p的胞外活性測試，尋找是否還有非組蛋白的功

能性受質存在。

The basic unit of chromatin consists of 146 bps of DNA

wrapped around a histone octamer, which is composed of two

copies of each of the four core histones: H2A, H2B, H3 and

H4. The covalent modification of core histones modulates

genome function by contributing additional epigenetic

information. At this stage, one of the common covalent

histone modification is methylation. In recent years ,

methylation is determined to be reversible and another

dynamic group protein covalent modification,

demethylation, is established. Therefore,the covalent

modification of histones plays a pivotal role in the

epigenetic control of gene expression.

In this study, substrate specificity of Saccharomyces

cerevisiae JHD1p demethylase is studied. Based on the

information known from literature, Saccharomyces

cerevisiae JHD1p demethylase is an enzyme

with activity in vitro, and most of the studies have

focused on the substrate role of histone proteins as the

main research goal. Nevertheless, this study would like to

use the in vitro activity of enzyme JHD1p to

find out whether there is any functional non-histone

substrates existing in Saccharomyces cerevisiae.

謝誌...................................................Ⅰ

中文摘要...............................................Ⅱ

英文摘要...............................................Ⅳ

縮寫檢索表.............................................Ⅵ

目錄...................................................Ⅶ

圖目錄.................................................Ⅹ

表目錄...............................................ⅩⅠ

第一章、緒論............................................1

(一)組蛋白去甲基化作用................................. 3

(二)研究動機........................................... 5

第二章、材料............................................6

(一)菌株................................................6

(二)實驗儀器............................................6

第三章、實驗方法........................................8

(一)構築啤酒酵母菌JHD1基因於大腸桿菌....................8

1.萃取酵母菌(S. cerevisiae) genomic DNA.................8

2.設計引子..............................................8

3.聚合酶鏈鎖反應........................................9

4.瓊脂膠體電泳..........................................9

5.純化聚合酶鏈鎖反應之產物.............................10

6.補A反應(在純化產物兩端加上adenosine).................11

7. TA cloning..........................................11

8.勝任細胞的製備.......................................12

9.轉型作用.............................................13

10.藍白篩選............................................13

11.質體萃取............................................13

12.限制酶酵素進行確認..................................14

13. DNA定序............................................15

(二) JHD1基因次選殖至pGEX-4T-1和pET28c表現載體上
(subcloning)...........................................15

1.酶切反應.............................................15

2.酶接反應.............................................16

3.確認基因接入表現載體.................................16

(三) JHD1p重組蛋白質表現與純化........................17

1.以E. coli菌種表現JHD1p重組蛋白質.....................17

2.超音波破菌...........................................17

3. GST-tag融合純化法純化重組蛋白.......................18

4. His-tag融合純化法純化重組蛋白.......................18

5.蛋白質序列鑑定(MALDI-TOF MS)....................19

(1)、膠體清洗.......................................19

(2)、還原與烷基化................................19

(3)、trypsin 處理................................20

(4)、胜肽萃取....................................20

(5)、質譜儀分析........................................20
.
(四)測定JHD1p重組蛋白之其去甲基酶活性及分析其受質......21

1.受質的製備...........................................21

2.去甲基酶反應活性測定.................................22

3.以SDS-PAGE初步測定去甲基酶活性.......................22

第四章、結果與討論.....................................24

1.蛋白質表現結果.......................................25

2.放射性底片結果.......................................26

3.JHD1p去甲基酶胞外活性................................26

第五章、結論...........................................27

第六章、參考文獻.......................................28

圖目錄

圖一、 經含有Taq DNA polymerase + Pfu DNA polymerase(16:1)
的PCR反應下所得的JHD1預期產物。...............32

圖二、 設計EcoRI/BamHI 和NdeI/SalI切位的JHD1目標基因接合pGEM® -T Easy Vector後，分別以EcoRI/NdeI enzyme確認。
..............................................33

圖三、 JHD1目標基因接上pGEX 4T-1 vector以EcoRI/BamHI enzyme確認。.................................................34

圖四、 JHD1目標基因接上pET28c vector以NdeI/SalI enzyme確認。 .................................................35

圖五、 JHD1定序結果....................................36

圖六、 pGEX-4T-1-JHD1- tRNA380 (稱TRGJ)與pGEX 4T-1-tRNA380 (稱TRG) 18℃ 誘導 40 小時。............................38

圖七、 利用GST-Tag resin純化JHD1p。....................39

圖八、 pET28c-JHD1-tRNA380 (稱TRHJ)與pET28c-tRNA380 (稱 TRH)培養於LB medium / D-sorbitol/betaine 18℃ 誘導 40 小時。...................................................40

圖九、 利用His-Tag resin純化JHD1p。....................41

圖十、 JHD1p enzyme反應前之SDS-PAGE膠片。..............43

圖十一、[H3]胞內放射性標定反應SDS-PAGE電泳圖(Benzonase同時反應處理)(壓片前)。....................................44

圖十二、附圖十一[H3]胞內放射性標定反應底片圖(Benzonase同時反應處理) (洗片後)。...................................45

表目錄

表一、引子設計。.......................................46

表二、聚合酶鏈鎖反應條件。.............................46

表三、放射性去甲基反應。...............................47

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