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標題: | 酵母必需基因中 Hsp90 可緩衝突變的遺傳篩選 A genetic screen for Hsp90 bufferable mutants in yeast essential genes |
作者: | 林克文 Nicholas Francis Hoeffner |
指導教授: | 呂俊毅 Jun-Yi Leu |
關鍵字: | 緩衝遺傳變異,CBF5,Hsp90, Genetic Buffering,CBF5,Hsp90, |
出版年 : | 2023 |
學位: | 碩士 |
摘要: | Hsp90 是一種分子伴侶蛋白,對大部分真核生物蛋白質組的數量有直接或間接的影響。過去觀察到 Hsp90 具有緩衝遺傳變異的能力,也就是說,它在細胞中的存在可以抵銷或減弱與基因變異或突變體相關聯的表現型強度。目前人們已提出了幾種機制來解釋這種現象,其中一些機制需要 Hsp90 的結合並穩定突變蛋白質,而其他機制則認為,由於Hsp90 下游因子導致突變蛋白質的數量增加,使得該蛋白質能夠容忍其活性小幅度的降低。Hsp90 的緩衝能力在進化中可能扮演著重要角色,因為它決定了哪些變異體將有助於人口的表型多樣性以及在什麼條件下發揮作用。為了評估它們的影響力和性質,需要更好地了解受 Hsp90 緩衝的變異體的普遍性和特徵,以及主要的緩衝機制。在這篇論文中,我們利用偽尿嘧啶合成酶 CBF5 作為目標基因,創建並利用基因篩檢以識別在酵母基因中具有 Hsp90 緩衝作用的 CBF5 點突變。我們將由易錯聚合酶鏈反應產生的 CBF5 突變基因庫轉化為條件性 CBF5 酵母抑制株,並通過 Tet-Off 啟動子來控制 Hsp90 的水平。我們在正常和降低 Hsp90條件下對突變體的適應性進行測試,並鑒定出幾種具有 Hsp90 緩衝表現型的突變體。此外,我們還開發了一種基於複製板和影像分析以快速識別這些酵母菌落表現型的方法。 Hsp90 is a molecular chaperone with a direct or indirect impact on the abundances of large portions of eukaryotic proteomes. Hsp90 has previously been observed to buffer genetic variation, meaning that its presence in the cell can nullify or reduce the strength of phenotypes associated with genetic variants or mutants. Several mechanisms have been proposed to explain this phenomenon. Some of these require that Hsp90 binds and stabilizes the mutant proteins while others posit that increases in the abundance of the protein resulting from factors downstream of Hsp90 bring that protein to a level such that it can tolerate small reductions in activity. Hsp90 bufferability may play a critical role in evolution as it decides which variants will contribute to the phenotypic diversity of a population and under which conditions. In order to assess the strength and the nature of their impact, a better understanding of the prevalence and characteristics of Hsp90-buffered variants as well as the predominant mechanism of buffering is necessary. Here, we establish and conduct a screen for identifying Hsp90-bufferable point mutations in yeast essential genes using CBF5, a pseudouridine synthase, as the target. We transformed an error-prone PCR generated CBF5 mutant library into a CBF5 conditional knockdown strain with Hsp90 levels controlled by the Tet-Off promoter. We assayed the fitness of mutants in normal and reduced-Hsp90 conditions and identified several mutants with Hsp90-bufferable phenotypes. Furthermore, we developed an approach for rapidly identifying yeast colonies with these phenotypes based on replica plating and image analysis. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/88743 |
DOI: | 10.6342/NTU202302603 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 基因體與系統生物學學位學程 |
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