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系統識別號 U0002-2707200917211100
中文論文名稱 酵母菌YJR129c合成致死基因的探討
英文論文名稱 Synthetic Lethal Screening for YJR129c Gene of Saccharomyces cerevisiae
校院名稱 淡江大學
系所名稱(中) 生命科學研究所碩士班
系所名稱(英) Graduate Institute of Life Sciences
學年度 97
學期 2
出版年 98
研究生中文姓名 陳國真
研究生英文姓名 Kuo-Chen Chen
電子信箱 shadowwall@gmail.com
學號 694290247
學位類別 碩士
語文別 英文
口試日期 2009-07-23
論文頁數 44頁
口試委員 指導教授-陳銘凱
委員-鄧述諄
委員-官宜靜
中文關鍵字 合成致死  S-腺苷甲硫胺酸  基因剔除 
英文關鍵字 Synthetic Lethal  S-adenosylmethionine  Gene knockout 
學科別分類 學科別醫學與生命科學生物學
中文摘要 蛋白質的甲基化是一種常見的轉譯後修飾,雖然截至目前已發現許多甲基酶以及被甲基化的位置,但仍有許多的甲基酶以及其作用位置仍未被找出。
啤酒酵母菌(Saccharomyces cerevisiae)中的YJR129c目前僅推測其可能是依賴S-腺核苷甲硫胺酸的甲基轉移酶,經由綠螢光蛋白可得知它存在於細胞質中,但其作用目標與相關機轉皆不知道。
為了研究YJR129c與其他基因之間的關聯性,本實驗選擇了合成致死篩選法(Synthetic lethal screening)來觀察基因層面的關聯性,以推測YJR129c的功能。此方法是藉由剔除YJR129c這非必須基因,並藉由隨機突變來觀察,哪些基因在YJR129c不存在時參與了補足它的功能,這些經由篩選得到的結果,將有助於推測YJR129c的真正功能,以及其在功能路徑裡所扮演的角色。
英文摘要 Protein methylation is a phenomenon of frequent occurrence. Althought a lot of methyltransferases and their substrates have been identify, but still many methyltransferases or substrates remain unknown.
The YJR129c in Saccharomyces cerevisiae was predicted to be encoding an S-adenosylmethionine-dependent methyltransferase ; green fluorescent protein (GFP)-fusion protein localizes to the cytoplasm, but its substrate and functional pathway are totally unknown.
To analyze the genetic interaction between YJR129c and the other genes, we have used synthetic lethal screening to reveal the role of YJR129c. We constructed a strain with deleted YJR129c, which is a nonessential gene, then randomly mutagenized this strain to find out which gene complements YJR129c. These observations will help us to reveal the function of YJR129c and the pathway in which YJR129c plays a role.
論文目次 TABLE OF CONTENTS
謝誌..........................................................................................................................I
中文摘要............................................................................................II
英文摘要...........................................................................................III
Abbreviations...................................................................................IV
1. Introduction
1.1. Methyltransferase.........................................................................................1
1.2. Synthetic Lethal...........................................................................................2
1.3. ade2/ade3 Red/White Colony Sectoring System.........................................2
2. Material and Methods
2.1. Strain
2.1.1. E. coil Strain.........................................................................................4
2.1.2. Yeast Strain...........................................................................................4
2.2. Plasmid.........................................................................................................4
2.3. Primer...........................................................................................................5
2.4. Media............................................................................................................7
2.5. Reagents List................................................................................................8
2.6. Construction of Rescue Plasmid
2.6.1. Clone the YJR129c by Polymerase Chain Reaction(PCR)..................9
2.6.2. Agarose Gel Electrophoresis................................................................9
2.6.3. Recovering DNA from Agarose Gel....................................................9
2.6.4. Addition of 3’-A Overhangs to PCR Product.....................................10
2.6.5. T&A Cloning......................................................................................10
2.6.6. Heat Shock Competent Cells Preparation (E. coli)............................10
2.6.7. Blue/White Selection by Heat Shock Transformation (E. coli).........11
2.6.8. Minipreparation of Plasmid DNA......................................................11
2.6.8.1. Plasmid Minipapration by Alkaline Lysis..................................11
2.6.8.2. Plasmid Minipapration by Purification Kit II............................12
2.6.9. DNA Sequencing................................................................................12
2.6.10. Restriction Enzyme Digest of DNA.................................................12
2.6.11. T4 Ligation.......................................................................................13
2.6.12. Restriction Enzyme Digest for Check Rescue Plasmid...................13
2.7. Disruption of YJR129c by PCR-Mediated One-Step Gene Replacement
2.7.1. Amplification of Homologous Recombination DNA........................14
2.7.2. Transformation of Yeast by the Lithium Acetate Method.................14
2.7.2.1. Yeast competent cell for Lithium Acetate Method.....................14
2.7.2.2. Transformation by Lithium Acetate Method..............................15
2.7.3. Yeast Transformation by Electroporation...........................................15
2.7.3.1. Yeast competent cell for Electroporation...................................15
2.7.3.2. Transformation by Electroporation............................................16
2.7.4. Yeast Genomic DNA : Glass-bead Preparation..................................16
2.7.5. Check the CHY125 ΔYJR129c Strain by PCR..................................17
2.8. Disruption of YJR129c by Tagged PCR-Mediate Recombination
2.8.1. Amplification of Homologous Recombination DNA........................18
2.8.2. Yeast Competent Cells for Electroporation........................................18
2.8.3. Yeast Transformation by Electroporation...........................................18
2.8.4. Preparation of Yeast Genomic DNA..................................................18
2.8.5. Check the CHY125 ΔYJR129c Strain by PCR..................................19
2.8.6. Pop-Out..............................................................................................19
2.8.7. Check the Pop-Out Strain by PCR.....................................................19
2.9. Construction of pRS315/YJR129c Plasmid
2.9.1. Minipreparation of Plasmid DNA......................................................20
2.9.2. Restriction Enzyme Digest of DNA...................................................20
2.9.3. T4 Ligation.........................................................................................20
2.9.4. Characterization of pRS315/YJR129c Transformants........................20
2.10. Synthetic Lethal
2.10.1. Transforme the Rescue Plasmid to CHY125 ΔYJR129c..................21
2.10.2. Check the Rescue Plasmid from CHY125 ΔYJR129c.....................21
2.10.3. Random Mutagenesis by EMS.........................................................21
2.10.4. Non-Sectoring Strain Assay.............................................................22
3. Results
3.1. YJR129c Clone Contain 500 up/downstram..............................................23
3.2. CHY125 ΔYJR129c Contain Trp1 Selection Marker……………........….23
3.3. Check the Correct CHY125 ΔYJR129c Strain……...………........………23
3.4. Isolation of Plasmid-dependent mutants....................................................24
4. Discussion
4.1. YJR129c Clone Contain 500 bp up/downstream maybe form a secondary
DNA-structures...........................................................................................24
4.2. The Length of DNA Can Affect Recombination Frequency.......................24
4.3. The Survival Ratio of Random Mutation maybe too high.........................26
4.4. Improve the Non-Sectoring Strain Assay...................................................26
5. Figure
Figure 1. YJR129c Cloned by PCR.....................................................................27
Figure 2. YJR129c TA-Cloning and pYT79 were digested by XmaI and BamHI
.............................................................................................................28
Figure 3. Use of Polymerase Chain Reaction Epitope Tagging to knockout
YJR129................................................................................................29
Figure 4. YJR129c Knockout by PCR-mediate One-step Homologous
Recombination......................................................................................30
Figure 5. YJR129c Knockout by PCR-mediate One-step Homologous
Recombination.....................................................................................31
Figure 6. Primers YJR129c Knockout A/B are Design from “YJR130c to
YJR129c (40bp) + Right” and “SNR3 to YJR129c (40bp) + Left.......32
Figure 7. BLAST the Primers “YJR130c to YJR129c (40bp) + Right” to
Saccharomyces cerevisiae....................................................................33
Figure 8. BLAST the Primers “SNR3 to YJR129c (40bp) + Left” to
Saccharomyces cerevisiae....................................................................34
Figure 9. The First Check of YJR129c unpopped-out Strain...............................35
Figure 10. The Secondary Check of YJR129c unpopped-out Strain...................36
Figure 11. The Secondary Check of YJR129c unpopped-out Strain...................37
Figure 12. The First Check of YJR129c popped-out Strain.................................38
Figure 13. The Secondary Check of YJR129c popped-out Strain.......................39
Figure 14. The Non-sectoring clones always contain several all white clones...40
6. Appendix..........................................................................................41
7. Reference..........................................................................................42
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