Yeast CSM2 is essential for meiotic DNA recombination and segregation

在酵母菌Saccharomyces cerevisiae進行減數分裂時，DNA重組會經由兩個不同的途徑產生基因互換或是非基因互換兩種產物。已知，利用基因晶片分析已知，在酵母菌進行減數分裂時Csm2之mRNA會被轉錄調控，在本篇論文中首先構築表現CSM2-ProA之菌株，利用免疫染色方法觀察其在細胞內之位置，及其在細胞分裂與減數分裂時期之蛋白表現。接著構築Csm2基因剔除之菌株觀察於Csm2基因缺陷時對粒線體能量代謝之影響，再構築另一Csm2基因剔除之菌株(YPH607)，其特徵為含一對染色體對，其上帶有人類著絲點及端粒基因序列，並可利用其上之營養篩選標誌來觀察在Csm2基因缺陷下染色體複製分離是否正常。最後利用分子生物學之方法分析減數分裂進行時Csm2對DNA重組及染色體分離的影響。我們觀察到與野生種比較之下，Csm2基因剔除並不會影響粒線體能量代謝途徑且 Csm2基因剔除並不影響有絲分裂之染色體分離，在減數分裂中Csm2基因剔除之基因互換的產物明顯下降，但不影響DSBs的發生，大多數的細胞會有停留在第一次減數分裂，這個發現代表Csm2在減數分裂中參與的時間點在SEIs之後，基因互換形成之前，其可能不參與有絲分裂同源染色體的分離。During meiosis in Saccharomyces cerevisiae, DNA recombination is essential for successful chromosome segregation to yield crossover and non crossover products. CSM2 is one of the yeast open reading frames (ORFs) that are transcriptionally induced in a meiosis-specific fashion. In this study, we first constructed a strain expressing CSM2-ProA to confirm its inner cell localization in mitotic cells and meiotic cells respectively. Then, Csm2 null mutant in SK1 background was constructed to determine of CSM2 affect mitochondrial metabolism pathway. Furthermore, Csm2 null mutant in YPH 607 background, carrying a pair of yeas artificial chromosomes YAC12-UT and YAC12LH was constructed and used to observe if the chromosome segregation is normal without CSM2. Finally, we use physical analysis of meiotic DNA recombination at HIS4LUE2 to analysis if csm2 null mutant can affect the meiotic DNA recombination mechanism. We get those results that (1) CSM2 does not affect mitochondrial energy metabolism pathway, (2) CSM2 neither affect chromosome segregation in mitotic cells, (3) In csm2 null mutant, DSBs formation and processing is normal, crossover product has a dramatically decrease, and most cells were arrested in MI, which indicated csm2 participate the events after SEIs before the crossover product formation during meiosis, and csm2 might not participate in mitotic homologous chromosome segregation events. Its role in mitosis still has to be investigated.中文摘要…………………………………………………………………..…………………. ..I Abstract…………………………………………………………………….………….……….II Index..................................................................................................................................…....IV List of Figures....................................................................................................................….....V List of Tables……………………………………………………….………………………….VI List of Abbreviations………………………………………………………………………....VII 1. Introduction.......................................................................................................................…..1 2. Material and Method...……………………………………………………………………...4 2-1. Plasmids...........................................……………….……….………….……………..…4 2-2. Yeast strains and culture techniques……………….……….………….……………..….4 2-3. Preparation of total cell lysates with trichloroacetic acid (TCA) precipitation method ……………………………….………………………………………………....5 2-4. Western blotting analysis and immunological reagents…………............……………....5 2-5. Mitotic YAC loss assay…………….…………………………………….……………..6 2-6. Preparation of yeast genomic DNA and Southern blotting……………….…………….6 2-7. Preparation of yeast nuclei fraction..................................................................................7 3. Result......................................................................................................................................9 3-1. Csm2-ProA expressed in both mitotic and meiotic cells……………….………….......9 3-2. CSM2 do not affect mitochondrial energy metabolism..................…….………...….....10 3-3. The localization of Csm2-ProA in meiotic time course .....................………...……….11 3-4. csm2 null mutant has no chromosome segregation defect in mitosis cells....……....….13 3-5. Physical analysis of meiotic DNA recombination of csm2 null mutant strain……...….14 4. Discussion….……………………………………………………………………….........…17 4-1. Functions of CSM2 in mitotic cells....................................................................….......17 4-2. Localization and function of Csm2 protein in meiotic cells........................…..……....18 5. Reference……………………………………………………………….………….…….…1