王廷方臺灣大學：生化科學研究所梁淑珊Liang, Shu-ShanShu-ShanLiang2007-11-262018-07-062007-11-262018-07-062005http://ntur.lib.ntu.edu.tw//handle/246246/52811中文摘要 Pep4蛋白是位於酵母菌液泡的天門冬胺酸水解Abstract Pep4 protein, a nonessential Saccharomyces cerevisiae vacuole aspartic protease, is indispensable for meiosis. Mutation or deletion of PEP4 gene leads to a cell cycle arrest before the first meiotic nuclear division. In addition, Pep4 protein and its proteolytic activity both are up-regulated during early meiosis. The latter has seriously impeded previous meiosis studies using biochemistry or proteomic approach. Hence, it is intriguing, probably also important, to understand the function of Pep4 protein during meiosis. Here we report that the pep4 mutant cell is defective in meiotic DNA recombination. Meiotic DNA recombination is initiated via formation of double strand breaks(DSBs), and subsequently produces recombinational products from the DNA of two parental homologous chromosomes. It had been shown previously that distribution of DSBs along the meiotic chromosomes is not random. Analysis of DSB hotspots revealed that the pep4 mutant produced normal levels of DSBs but almost no final recombination products. Intriguingly, the distribution of DSBs along meiotic chromosomes in the pep4 mutant was different from that of wild type cell. Our studies also indicated that several chromosome proteins were not properly degraded in the pep4 mutant during meiosis. We proposed that accumulation of superfluous chromosome proteins might influence structure of meiotic chromosomes and subsequently alter the chromosome distribution of DSBs.中文摘要 1 Abstract英文摘要 2 目錄 3 縮寫表 4 第一章 序論…………………………………………………………………………..6 1.1減數分裂的重要性 6 1.2酵母菌Pep4蛋白質是正常進行減數分裂不可或缺的 7 1.3 pep4突變株不能進行減數分裂第一次染色體分離 7 1.4 減數分裂DNA重組機制與研究方法介紹 8 1.5減數分裂染色體型態變化 9 1.6減數分裂DNA重組發生受染色體結構影響 10 第二章 材料與方法…………………………………………………………………13 2.1酵母菌基因轉型 13 2.2酵母菌減數分裂細胞週期的同步化 13 2.3螢光激活細胞分類儀(Fluorescence Activated Cell Sorter)分析DNA複製 14 2.4細胞核散灑（Nuclear spreading） 14 2.5酵母菌染色體免疫螢光染色法 15 2.6 抽取酵母菌DNA 15 2.7南方印跡術(southern blot)與DNA雜交 (hybridizaiton)實驗 16 2.8三氯乙酸（TCA）蛋白質沉澱法製備酵母菌全細胞蛋白樣品 17 2.9 蛋白質電泳與西方印跡術(Western blot) 18 2.10 脈衝式電泳（Pulsed field gel electrophoresis） 18 2.11實驗菌株 20 2.12實驗培養基 21 第三章 結果…………………………………………………………………………22 3.1建立並確認PEP4基因剔除的酵母菌菌株 22 3.2分析pep4與其他基因共同剔除時染色體分離狀況 22 3.3 pep4菌株雙股斷裂發生情形與野生菌株有明顯不同 24 3.4利用Dmc1免疫螢光方法檢測pep4菌株雙股斷裂的發生情形 25 3.5 pep4菌株形成正常的聯會複合體 26 3.6利用脈衝式電泳分析pep4菌株內雙股斷裂發生情形 26 第四章 討論…………………………………………………………………………28 4.1pep4突變株可以產生雙股斷裂 28 4.2 pep4 突變株部分雙股斷裂位置改變 28 4.3 雙股斷裂無法修復完成重組的原因 29 第五章 結果與圖表………………………………………………………………. 30 圖一、酵母菌減數分裂染色體的重組 30 圖二、第三條染色體雙股斷裂位置分布不均勻 31 圖三、分析減數分裂HIS4LEU2 DNA重組與雙股斷裂 32 圖四、pep4菌株蛋白質分解情況 33 圖五、Mcd1與Rec8蛋白在減數分裂分解情形 34 圖六、HIS4LEU2雙股斷裂累積的狀況 35 圖七、偵測YCR048與GAT1雙股斷裂的形成 36 圖八、pep4不影響histone3 Lys4的甲基化與其跟DNA作用的能力 37 圖九、pep4可以形成Dmc1蛋白小點 38 圖十、pep4可形成完整的聯會複合體 39 圖十一、利用PFGE分離並偵測dmc1與dmc1pep4 雙股斷裂的產生 40 圖十二、pep4造成染色體結構改變的假說 41 表格一、分析pep4與其他雙突變株的DNA分離狀態 42 第六章 附錄………………………………………………………………………..43 附一、利用南方氏印跡術確認dmc1pep4雙剔除 44 附二、利用南方氏印跡術確認spo13與mnd2剔除 45 第七章 參考文獻…………………………………………………………………...451913792 bytesapplication/pdfen-US減數分裂酵母菌重組meiosisyeastrecombinationotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/52811/1/ntu-94-R92b46039-1.pdf