酵母菌Ssa3蛋白質於減數分裂時期功能之研究

Abstract

老鼠熱休克蛋白質HSP70-2（70-kDa heat shock protein, HSP70）已知在減數分裂過程中扮演重要的角色，而酵母菌（budding yeast, Saccharomyces cerevisiae）中和老鼠HSP70-2蛋白質最為相似的是Ssa3（stress-seventy subfamily A）蛋白質，但其確切的功能目前還不清楚。本論文主要的目標在於研究酵母菌Ssa3蛋白質於減數分裂時期的功能。 首先，我們利用分子遺傳學方法獲得一些SSA3基因完全破壞的突變株（null mutants），結果發現ssa3突變本身對正常細胞的減數分裂沒有明顯的效應，但卻對zip1突變株的缺失檢控有影響。Zip1是酵母菌聯會複合體（synaptonemal complex, SC）中央區域的重要結構蛋白，在zip1突變株中，同源染色體無法聯會，細胞週期會停止（arrest）在粗絲期（pachytene）無法繼續完成減數分裂產生孢子。若在zip1突變株中將SSA3基因破壞，卻可以隱抑（suppress）zip1的缺失，使zip1突變株略過（bypass）粗絲期檢控點繼續進行減數分裂，但zip1ssa3的孢子存活率明顯比野生型低，顯示ssa3隱抑zip1不是透過修補zip1的缺失，而是ssa3在zip1中會影響粗絲期檢控點的作用。而ssa3突變無法使其他停止在粗絲期的突變株，如hop2或dmc1產生孢子。 利用蛋白質免疫轉印技術（Western blot analysis）分析Ssa3蛋白質，我們觀察到Ssa3蛋白質不只在減數分裂中才表現，但其表現量會隨著減數分裂的進行而增加。免疫螢光分析的結果顯示，不論是野生型或是zip1突變株，Ssa3蛋白質都同時存在於細胞核以及細胞質中；更進一步的觀察減數分裂粗絲期的染色體，發現Ssa3蛋白質位於野生型及zip1突變株的粗絲期染色體上，並且呈不完全連續的分佈。綜合以上的結果，我們推論在酵母菌進行減數分裂的過程中，Ssa3蛋白質可能參與維持染色體的適當構造（chromosome context），以使粗絲期檢控點能正確的作用。

The SSA3 (stress-seventy subfamily A) gene of Saccharomyces cerevisiae is a member of the HSP70 multigene family. To investigate the role of Ssa3 in meiosis, null mutants of SSA3 were generated and analyzed. ssa3 mutants undergo nearly wild-type levels of nuclear division and spore formation. Thus, Ssa3 is not required for sporulation in wild-type cells. However, ssa3 mutations can partially suppress a pachytene-arrest mutant, zip1, which has defects in chromosome structure and recombination. The spore viability of zip1ssa3 is about 41.6% of that of wild-type, suggesting that the recombination defects of zip1 persist in zip1ssa3 double mutants. These results indicate that ssa3 mutation might affect the normal function of the pachytene checkpoint in zip1 cells. Unlike zip1ssa3, ssa3 mutations fail to bypass other two pachytene-arrest mutants, hop2 and dmc1. Using epitope tagging strategy, the Ssa3 protein was detected by anti-HA antibodies. The Ssa3 protein is not a meiosis-specific protein, but its expression is slightly increased in meiosis. Chromosome spreading analyses indicate that Ssa3 protein is localized to pachytene chromosomes in both wild-type and the zip1 mutants. According to these results, we suggest that the Ssa3 protein may be involved in maintaining a proper chromosome structure for pachytene checkpoint signaling in budding yeast.