臺灣博碩士論文加值系統

減數分裂是真核生物進行有性生殖時特化的細胞週期，主要功能再促進親代染色體間發生DNA重組互換，並確保染色體正確分離產生染色體數目減半的配子細胞（如精子與卵）。為了達到此目的，在減數分裂的前期中段染色體會形成聯會絲複合（synaptonemal complex），使來自父母雙方同源染色體之間緊密配對，促進DNA重組發生於同源染色體間。聯會絲複合體迄今已發現剛好五十年，但其形成的機制還不清楚，是有性生殖研究最重要課題之一，本論文的研究結果，對此課題有開創性的新發現。生物學界最常用來研究聯會絲複合體形成機制的生物模式是酵母菌，其Zip1蛋白質是建構聯會絲複合體的主要結構蛋白，Zip1也在減數分裂前期初段時，負責將兩對非同源染色體著絲點間偶合在一起，此機制與同源染色體間DNA重組並無直接的關聯性，到了減數分裂前期中段時，減數分裂專一性蛋白質Zip3負責啟動聯會絲複合體的形成。本論文證明Zip3是類泛素（Smt3）的E3接合酵素，負責在減數分裂前期中段時，將染色體上的結構蛋白質（如Red1）進行類泛素修飾。我們也發現Zip1能結合被類泛素化修飾的染色體結構蛋白質（如Top2），在減數分裂前期初期負責非同源染色體著絲點間的偶合，然後在減數分裂前期中段促進聯會絲複合體的形成。

Synaptonemal complex （SC） is a proteinaceous complex that apparently mediates synapsis between homologous chromosomes during meiotic prophase. In S. cerevisiae, the Zip1 protein is the integral component of SC. In the absence of a DNA double-strand break or the SC initiation protein Zip3, Zip1 proteins aggregate to form a polycomplex （PC）. In addition, Zip1 is also responsible for DSB-independent non-homologous centromere coupling at early meiotic prophase. We report here that Zip3 is a SUMO E3 ligase and that Zip1 is a binding protein for SUMO-conjugated products. Our results also suggest that at early meiotic prophase, Zip1 interacts with Zip3-independent Smt3 conjugates （e.g., Top2） to promote non-homologous centromere coupling. At and after mid-prophase, the Zip1 protein begins to associate with Zip3-dependent Smt3 conjugates （e.g., Red1） along meiotic chromosomes in the wild-type cell to form SCs and with Smt3 polymeric chains in the zip3 mutant to form PCs.

口試委員會審定書
誌謝
中文摘要 5
英文摘要 Abstract 6
縮寫 7
第一章 序論
1.1 減數分裂的重要性 9
1.2 減數分裂DNA重組機制 9
1.3 減數分裂染色體型態變化 11
1.4 sumoylation對細胞的影響 14
第二章 材料與方法
2.1 酵母菌基因轉型 17
2.2酵母菌減數分裂細胞週期的同步化和孢子形成機率計算 18
2.3三氯乙酸蛋白質沉澱法製備酵母菌全細胞蛋白樣品 19
2.4蛋白質電泳與西方印跡術 19
2.5酵母菌雙雜合分析 20
2.6大腸桿菌重組蛋白質純化 21
2.7酵母菌重組蛋白質純化 24
2.8 In vitro 類泛素多聚合體分析 26
2.9 去磷酸化分析 27
2.10 Zip1C’末端接合分析 27
2.11實驗菌株 29
2.12實驗載體 33
2.13實驗培養基 36
第三章 結果
3.1 zip3突變菌株進入減數分裂時，在前期中段時期，類泛
素會形成多聚合體（Smt3 polymeric chain）。 38
3.2 Zip3是類泛素接合酶的新成員。 39
3.3 Zip1會與類泛素化產物結合。 42
第四章 討論 45
第五章 結果與圖表
圖一 觀察野生型和ulp2突變株在減數分裂時類泛素化
蛋白質的表現 47
圖二 觀察野生型和zip1-3突變株在減數分裂時類泛素化
蛋白質的表現 48
圖三 離胺酸突變類泛素在zip3突變菌株行減數分裂時的
蛋白表現 49
圖四 Zip3是類泛素接合酶的新成員 50
圖五 Zip3具有類泛素接合酶的活性 52
圖六 類泛素多元聚合體形成需要Zip1蛋白質 54
圖七 Zip1蛋白質C’末端具有類泛素結合模體 55
圖八 Zip1蛋白質會和被類泛素化的產物結合 56
表一 Zip3會專一性的與Ubc9和Smt3結合 51
第六章 參考文獻 57
第七章 附錄
附錄一
SUMO Modifications Control Assembly of Synaptonemal Complex and
Polycomplex in Meiosis of Saccharomyces cerevisiae
Cheng and Lo, et al. Gene and Development, 2006
附錄二
SUMO-mediated regulation of synaptonemal complex formation during meiosis. Carlos Egydio dde Carvalho and Monica P. Colaiacovo, 2006

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