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  • 學位論文

酵母菌YPL014W蛋白之功能特徵分析

Functional characterization of yeast YPL014W

指導教授 : 鄧述諄

摘要


在大部分的真核生物裡,端粒的複製是藉由端粒酵素(telomerase)維持。缺乏端粒酵素的酵母菌族群仍有一些倖存的細胞,能夠利用DNA重組(recombination)的方式有效的延長端粒長度。第二類型的倖存細胞利用telomerase-independent alternative lengthening of telomeres (ALT) pathway的方式維持端粒的功能,而YPL014W在酵母菌細胞進入ALT pathway時表現量上升。由於已知Ypl014w可與Cdc28、Cln3等細胞週期調節蛋白形成複合體 (complex)(Uetz et al., 2000),顯示Ypl014w可能參與細胞週期的調控路徑,又因其在ALT pathway中表現量上升,這暗示端粒延長路徑與細胞週期調控之間的關聯。因此本實驗藉由觀察Ypl014w對Cdc28、Cln3的影響,來推知其功能,目前已知YPL014W的剔除不會影響Cdc28-Cln3複合體對下游蛋白質Whi5的磷酸化,也不會影響細胞週期的進行;另外我們發現Ypl014w為一可被磷酸化之蛋白質,但CLN3的剔除和CDC28的溫度敏感突變株同樣不會影響Ypl014w的磷酸化。另外藉由synthetic lethal method沒有找到YPL014W的redundant gene。進一步利用yeast two hybrid確認Cdc28、Cln3和Ypl014w的結合時,發現Cdc28和Ypl014w沒有直接的結合關係,而Cln3的部分則無法證明,同時也利用Ypl014w做為bait,以酵母菌雙雜交實驗尋找和它結合的蛋白質,還有進行IP,利用SDS-PAGE分開後,解Mass以尋找和Ypl014w有結合的蛋白質,但都沒有所獲。 另一方面之前的實驗證實YPL014W的表現是受到Mcm1所調控,Mcm1是一個調控許多細胞週期相關基因的轉錄因子,在這裡藉由chromatin IP直接證明了Mcm1會結合到YPL014W的啟動子上,且證明了之前所預測的兩個ECB elements的確是Mcm1的結合處。

關鍵字

YPL014W

並列摘要


Telomere is replicated by telomerase in most eucaryotes. Most yeast cells that lack telomerase enter into senescence and eventually die. However, a few cells bypass the senescence phenotype and survive. These survivors elongate their telomeres by the alternative recombination pathway. The transcriptional level of YPL014W is increased in telomerase-independent alternative lengthening of telomeres (ALT) pathway, which is used by type II survivor cells. Previous study showed that Ypl014w forms a complex with Cdc28 and Cln3(Uetz et al., 2000), indicating that Ypl014w may involve in cell cycle regulation. Study the function of Ypl014w may help us to find the relationship between telomere elongation pathway and cell cycle regulation. In my study, YPL014W knockout has no influence on cell cycle regulation and the phosphorylation status of Cdc28-Cln3 substrate Whi5. I also find that Ypl014w is a phosphorylated protein, but CLN3 knockout and temperature sensitive mutant of CDC28 have no influence on its phosphorylation status. I did not find the redundant gene of YPL014W with synthetic lethal. I can not confirm the previous study that Ypl014w interacts with Cdc28 and Cln3 in yeast two hybrid. I did not find other proteins that interact with Ypl014w in yeast-two-hybrid screen and IP analysis. According to sequence analysis, YPL014W expression may be controlled by Mcm1, a transcription factor that regulates many cell cycle regulated genes. I showed that Mcm1 binds to the promoter of YPL014W with CHIP and found the two ECB elements that Mcm1 binds.

並列關鍵字

YPL014W

參考文獻


Teng, S. C., Chang, J., McCowan, B., and Zakian, V. A. (2000).
Abraham, D. S., and Vershon, A. K. (2005). N-terminal arm of Mcm1 is required for transcription of a subset of genes involved in maintenance of the cell wall. Eukaryot Cell 4, 1808-1819.
Chang, V. K., Donato, J. J., Chan, C. S., and Tye, B. K. (2004). Mcm1 promotes replication initiation by binding specific elements at replication origins. Mol Cell Biol 24, 6514-6524.
Chen, Q., Ijpma, A., and Greider, C. W. (2001). Two survivor pathways that allow growth in the absence of telomerase are generated by distinct telomere recombination events. Mol Cell Biol 21, 1819-1827.
Costanzo, M., Nishikawa, J. L., Tang, X., Millman, J. S., Schub, O., Breitkreuz, K., Dewar, D., Rupes, I., Andrews, B., and Tyers, M. (2004). CDK activity antagonizes Whi5, an inhibitor of G1/S transcription in yeast. Cell 117, 899-913.

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