臺灣博碩士論文加值系統

生物細胞中的核酸配對錯誤，對生物體所造成的影響十分重大。基因層次的錯誤配對會造成基因突變甚至癌症的發生。目前大腸桿菌中之MMR( mismatch repair )系統以Mut HLS pathway的研究最為透徹。而在真核細胞中，核酸配對錯誤由MSH2與MSH6形成複合體MutSα進行辨識結合；而MSH2與MSH3形成之複合體MutSβ則對嵌入或刪除寡核甘酸具有結合辨識的能力。
本論文以斑馬魚為材料，因其有容易在實驗室繁殖、魚卵呈透明及4天即可由胚胎發育至可游動之魚苗等許多優點，故常被用來做胚胎發育及遺傳學研究。我們首先依據各物種的MSH2保守區設計一組專一性引子，得到斑馬魚中MSH2 cDNA約2 kb片段，進行5’- RACE及3’- RACE，獲得斑馬魚MSH2之cDNA全長序列。經全長PCR及定序後，發現其ORF (open reading frame) 含有2811個nucleotides，可轉譯936個胺基酸，與人、老鼠MSH2進行比對後其相似度高達69 %，可得知為斑馬魚MSH2基因，而與斑馬魚MSH6基因比對相似度雖只有40%，但兩者皆有Mismatch recognition region與ATP-binding sites類似區域，其為MutS Homolog之特色。接著利用TNT® in vitro transcription and translation套組進行蛋白質表現，以SDS-PAGE分析後獲得分子量約為105 kDa之MSH2，由親和性吸附實驗發現其對GT 錯誤配對比正常配對為有較強之辨識能力。另一方面以北方點墨法及半定量PCR發現12至84小時MSH2與MSH6之mRNA 表現量有隨著胚胎發育而減少之情形，而由凝膠阻滯試驗顯示84小時胚胎粗萃取物其辨識能力很強，可與G-G與G-T異雙股DNA形成明顯之複合物，表示此發育時期之斑馬魚並不因MSH2與MSH6之mRNA 表現量減少而影響其辨識能力。熱休克 ( 37℃/30 min ) 處理後之36與84小時斑馬魚，發現MSH2與MSH6 mRNA表現量有減少之情形而隨著溫度回降以及時間增加後有緩慢上升的現象，此現象又以84小時斑馬魚MSH2與MSH6之 mRNA較為明顯，而熱休克並未對β- actin產生影響，因此可知熱休克會專一性地影響MSH2與MSH6 mRNA的表現，而其mRNA的表現下降是因其生成被抑制或因不穩定而被降解，其確實原因尚待實驗研究證明。

Mismatched nucleotides in eukaryotic DNA are recognized by heterodimeric complexes of proteins homologus to the bacterial MutS protein. We attempted to study gene expression of MutS homologues in developing vertebrates using zebrafish ( Danio rerio ) as a model system. A novel cDNA encoding a zebrafish MutS homologue, zfMSH2, was obtained by reverse transcription followed by rapid amplification of cDNA ends ( RACE ). The zfMSH2 cDNA has an open reading frame of 2811 nucleotides that can be translated into a polypeptide of 936 amino acids with a molecular mass about 105 kDa. The amino acid sequence of zfMSH2 shares 72 %、69 % and 69 % identity to African clawed frog 、human and mouse MSH2, respectively.
The zfMSH2 protein contains four ATP-binding motifs and a mismatch recognition region conserved among MutS homologues. Recombinant zfMSH2 obtained from in vitro transcription/translation showed a preferential binding to a heteroduplex containing a G-T mismatch as shown by affinity adsorption.
Northern blot analysis detected apparent levels of zfMSH2 and zfMSH6 mRNA expressions in 12 and 36-hr-old zebrafish embryos, while these expressions are significantly reduced in 60-hr-old late embryos and 84-hr-old larvae, indicating a stage-dependent regulation of MSH2 and MSH6 expression in developing zebrafish.
Incubation of G-T and G-G heteroduplex probes with the extracts of 12 to 60-hr-old embryos generated predominantly high-shifting binding complexes with similar band intensities. Although low in zfMSH2 and zfMSH6 productions, mismatch recognition proteins contained in 84-hr-old zebrafish bound apparently stronger to a G-T than to a G-G heteroduplex probe, producing both high and low-shifting retardation complexes. The strong G-T complexes generated by 84-hr-old zebrafish extracts might result from the binding of G-T recognition proteins specifically produced in more mature zebrafish to mismatched DNA.
The expressions of zfMSH2 and zfMSH6 mRNA in 36 and 84-hr-old zebrafish were sensitive to heat stress at 37 ℃ for 30 min, as a reduction in the levels of these MSH mRNA species were observed immediately after heat treatment. The expressions of zfMSH2 and zfMSH6 mRNA returned to normal levels about 2hrs after incubation at 28.5 ℃. Heat stress did not affect the expression of β-actin mRNA in developing zebrafish at all. Whether heat shock inhibited the transcription of zfMSH2 and zfMSH6 genes or caused an accelerated mRNA degradation remain to be determined.

總目次
總目次……………………………………………………………………i
圖目次…………………………………………………………………..iii
表目次…………………………………………………………………..iii
誌謝……………………………………………………………………..iv
縮寫表…………………………………………………………………...v
英文摘要.………………………………………………………………vii
中文摘要………………………………………………………………..ix
壹、序論…………………………………………………………………1
貳、實驗材料與方法……………………………………………………6
一、材料………………………………………………………………6
二、方法……………………………………………………………..17
(一) 斑馬魚之錯誤配對辨識蛋白MSH2 cDNA的選殖……...17
1. 斑馬魚之飼養………………………………………….17 2. 斑馬魚錯誤配對辨識蛋白MSH2基因專一性寡
核苷酸引子之設計……………………………………..17
3. 斑馬魚總量 RNA之抽取及定量…………………………18
4. 反轉錄聚合酶連鎖反應………………………………..19
5. 電泳分析膠體之製備及電泳分析………………………..20
6. DNA分子之萃取………………………………………21
7. 選殖………………………………………………………..22
8. Rapid Amplification of cDNA Ends ( RACE )……………23
(二) 斑馬魚之錯誤配對辨識蛋白MSH2 cDNA之表現……...26
1. 斑馬魚MSH2基因全長表現型載體之建構與分析…….26
2. 蛋白表現與樣品分析………………………………….….29
3. 活性分析…………………………………………………..31
(三) 斑馬魚胚胎時期之錯誤配對辨識蛋白MSH2表現
與熱休克之影響…………………………………………..33
1. 斑馬魚胚胎之收集及熱休克之處理…………………….33
2. 斑馬魚胚胎時期總量 RNA之抽取及定量……………...34
3. 半定量反轉錄聚合酶連鎖反應……………………….35
4. 北方點墨法…………………………………………….37
5. 胚胎細胞萃取液之製備……………………………….…41
6. 蛋白質定量法…………………………………………….41
7. 放射性錯誤配對雙股DNA的製備……………………...42
8. 凝膠阻滯試驗……………………………………………..44
參、結果………………………………………………………………..45
一、斑馬魚錯誤配對辨識蛋白MSH2 cDNA之選殖…………45
二、班馬魚之錯誤配對辨識蛋白MSH2 cDNA之表現
與活性分析……………………………………………...….46
三、斑馬魚胚胎發育時期MSH2及MSH6之表現與
錯誤配對辨識活性檢測…………………………………….47
四、熱休克對斑馬魚胚胎時期 MSH2及MSH6表現
之影響……………………………………………………….49
肆、討論………………………………………………………………..51
伍、參考文獻…………………………………………………………..53
陸、附圖………………………………………………………………..60
柒、附表………………………………………………………………..81
圖目次
附圖一……………………………………………………………..……60
附圖二………………………………………………………………..…61
附圖三………………………………………………………………..…62
附圖四………………………………………………………………..…63
附圖五………………………………………………………………..…64
附圖六…………………………………………………………………..65
附圖七………………………………………………………………..…66
附圖八………………………………………………………………..…67
附圖九………………………………………………………………..…70
附圖十………………………………………………………………..…72
附圖十一……………………………………………………………..…73
附圖十二………………………………………………………..………74
附圖十三………………………………………………………..………75
附圖十四………………………………………………………..………76
附圖十五………………………………………………………..………77
附圖十六………………………………………………………..………78
附圖十七…………………………………………………………..……79
附圖十八…………………………………………………………..……80
表目次
附表一……………………………………………………………..……81
附表二………………………………………………………………..…82
附表三………………………………………………………………..…83

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施錦毅，斑馬魚胚胎中DNA結構變異辨識蛋白之檢測與分離，國立臺灣海洋大學水產生物技術研究所碩士論文，九十一年六月。
閻惠玲，斑馬魚核酸配對錯誤修補辨識蛋白MSH6之分子選殖及表現，國立臺灣海洋大學水產生物技術研究所碩士論文，九十年六月。