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研究生:賴怡如
研究生(外文):LAI, YI-JU
論文名稱:酵母菌外側要素蛋白Red1磷酸化的訊息調控以及Red1磷酸化在減數分裂的功能
論文名稱(外文):Genetic requirements and meiotic function of phosphorylation of the yeast axial element protein Red1
指導教授:王廷方
指導教授(外文):Wang, Ting-Fang
口試委員:王廷方薛一蘋王群鄭明媛陳瑞祥  
口試委員(外文):Wang, Ting-FangHsueh, Yi-PingChung WangMing-yuan ChengRuey-Shyang Chen
口試日期:2011-01-25
學位類別:博士
校院名稱:國防醫學院
系所名稱:生命科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:英文
論文頁數:59
中文關鍵詞:減數分裂磷酸化聯會複合體同源重組
外文關鍵詞:Red1Synaptonemal complexLateral elementphosphorylationMeiosis
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減數分裂是真核生物進行有性生殖時特化的細胞週期,主要的功能在產生染色體數目減半的配子,譬如:哺乳動物的精子、卵以及酵母菌的孢子等。聯會複合體(synaptonemal complex)是減數分裂特有的三層蛋白結構,主要功能在連接配對的同源染色體以及促進同源染色體間的重組。同源重組確保同源染色體正確分離進而產生存活且有功能的孢子。聯會複合體包括一層中心要素(central element)和兩層外側要素(lateral element)。Red1, Hop1和Mek1是聯會複合體外側要素的組成蛋白。過去的科學家認為,Red1的磷酸化在減數分裂扮演重要的角色,它能夠傳導DNA損傷的訊息以及避免聯會複合體崩解。在此,我們利用改良過的蛋白質萃取技術發現Red1蛋白的磷酸化主要是由兩個訊息傳導路徑所調控,其中一條與Cdc28相關,另一條與Cdc28無關。而在 Cdc28無關的訊息傳導路徑,需要Hop1,以及Red1、小泛類素蛋白鏈 (SUMO chain) 的聯繫作用。和過去的研究結果相反,我們發現,Red1磷酸化與DNA損傷偵測蛋白Mec1,Tel1、DNA雙股螺旋斷裂以及Mek1磷酸脢無關。為了瞭解Red1磷酸化的功能,我們利用區域交換策略(domain swap approach)找到了一個沒有磷酸化修飾的Red1突變株:Red114A。 RED114A突變株形成孢子的比例以及孢子存活率和野生型相同。這些證據顯示,Red1的磷酸化並不影響酵母菌孢子的存活率。本研究有兩個重點:第一,我們發現Red1的磷酸化不影響正常孢子的形成。第二,我們為酵母菌減數分裂領域建立了一個有效萃取蛋白、避免大量蛋白質降解的標準流程。
Meiosis is a specialized process that produces haploid gametes from diploid precursor cells. The synaptonemal complex (SC) is a meiosis-specific tripartite proteinaceous structure that forms between two homologous chromosomes and promotes homologous recombination. Homologous recombination is required for proper segregation of homologous chromosomes to produce functional spores. The SC consists of one central element and two lateral elements. Red1, Hop1 and Mek1 are meiosis-specific proteins located at the lateral elements. It was reported that Mek1-dependent Red1phosphorylation plays important roles in transduction of meiotic DNA damage signal and in prevention of the exit from the pachytene chromosomes. By using a modified protein extraction method, we discovered that two pathways responsible for Red1 phosphorylation: a Cdc28-dependent and a Cdc28–independent pathway. Cdc28-independent Red1 phosphorylation depends on Hop1 and interaction of Red1/SUMO-chain. In contrast to previous studies, our study demonstrates that Red1 phosphorylation is independent of DSB formation, Mec1/Tel1 DNA damage checkpoint kinases and Mek1. By using a domain swap approach, we have identified a non-phosphorylated Red1 mutant: Red114A. RED114A has a sporulation efficiency and spore viability as high as the wild type, suggesting that Red1 phosphorylation is not required for functional spore production. In addition, RED114A shows no defect in DNA damage signaling. There are two points of this study: One is that Red1 phosphorylation is not essential for production of functional spores. The other is that we have established a protocol for efficient extraction of proteins from spores that avoids widespread proteolysis.
Introduction 1
Materials and methods 5
Yeast strains, media, western blot analysis, and custom gene synthesis. 6
Inhibition of the analogue-sensitive cdc28-as1 mutant. 6
Preparation of total yeast cell extracts. 6
Dephosphorylation assay. 6
Phos-tag affinity method. 7
Fluorescence-activated cell sorting (FACS). 8
Results 9
Red1 dephosphorylation is not required for exit from pachytene. 10
Loss of Cdc28/Clb5,6 or Ime2 kinase activity leads to Red1 hypophosphorylation and ubiquitination. 11
Dual modes of Red1 phosphorylation. 14
Cdc7 and the DNA damage checkpoint kinases are not involved in CDC28-independent Red1 phosphorylation. 19
Hop1 is required for CDC28-independent Red1 phosphorylation. 21
V5-Red114A is not phosphorylated in the absence of PP4 or when Glc7 is downregulated. 34
Discussion 39
Conclusion 44
Reference 47
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