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研究生:謝侑穎
研究生(外文):Yu-Ying Hsieh
論文名稱:探索熱休克蛋白九十在酵母菌系統之遺傳緩衝效應
論文名稱(外文):Exploring the genetic architecture buffered by Hsp90 in the yeast genome
指導教授:呂俊毅呂俊毅引用關係
指導教授(外文):Jun-Yi Leu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生命科學暨基因體科學研究所
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:2009
畢業學年度:97
語文別:英文
論文頁數:49
中文關鍵詞:遺傳緩衝
外文關鍵詞:genetic buffering
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基因型的改變導致表現型的改變是遺傳學的基本原則;然而,在生物系統中的某些生理機轉能夠隱藏基因型的變異、維持表現型的穩定,這樣的生命現象稱為遺傳緩衝效應。由於某些不適於原始環境但目前受隱藏的基因型變異可能促進生物在未知環境的演化,因此遺傳緩衝效應在生物演化上的角色不容小覷。從果蠅以及植物的研究發現,剔除熱休克蛋白九十能顯露不同遺傳品系的基因型變異;在酵母菌系統,剔除熱休克蛋白九十增加個體面臨遺傳干擾時的生長差異,故此蛋白質被認為參與遺傳緩衝效應。但是至今科學家們仍不了解熱休克蛋白九十如何居中協調遺傳緩衝效應,我的研究試圖了解哪些基因型變異的影響易受熱休克蛋白質所緩衝,以及熱休克蛋白質九十的表現量與緩衝能力之間的關聯性。
我在不同品系的酵母菌改變熱休克蛋白九十的表現量,觀察不同的遺傳因子決定表現型差異是否受到影響。整體基因表現概況是可被量化且能代表細胞生理變化的指標,因此在本研究中基因表現代表著所觀察的表現型。目前已在五種酵母菌品系(SK1, M1-2, M2-8, W303與S288C)中製備熱休克蛋白九十表現量不同的細胞。S288C品系被視為參考品系,其餘四種品系的整體基因表現與之比較。與參考品系比較後,某些基因在兩個酵母菌品系的表現差異隨著熱休克蛋白質九十表現量而有所變化,它們可能反應熱休克蛋白九十對於不同遺傳背景的緩衝結果,因而此類基因為本研究中所感興趣,暫且稱其為遺傳緩衝現象之報導基因。目前已篩選了三類的報導基因,大部分都具有專一於特定遺傳背景的特徵;此外,有些基因集中於特定的細胞路徑。然而,這些報導基因並無顯著地分布於熱休克蛋白九十交互作用的基因,故進一步的遺傳實驗能揭露影響報導基因表現的遺傳因子,以及透過生物資訊分析可幫助推斷熱休克蛋白九十與報導基因之間的關係。
為了瞭解遺傳變異受到不同程度的緩衝是否可能造成迥異的演化途徑,我將不同熱休克蛋白九十表現量的酵母菌細胞隨機累積基因突變達二千五百代,以觀察其中差異。藉由隨機選取單一菌落並劃至新的培養基,大部分的基因突變主要受隨機的力量被保留至下一代,故可針對較多樣的基因突變研究。整體來說,演化後的子代生長速率因累積的突變而比祖先細胞低,且具有非整倍體的特徵。在不同熱休克蛋白九十表現量的酵母菌細胞是否各具其特殊的染色體重組模式、或者其生長速率的變化反應出受熱休克蛋白九十緩衝的遺傳變異之作用,是下一步探討的議題。
Genetic dogma asserts that genetic change results in a change in phenotype. Genetic buffering is the surprising ability of an organism to maintain a stable phenotype despite the accumulation of genetic mutations. This “hidden” genetic variation is widely speculated to act as a reservoir in salutatory evolution and facilitate adaptation to novel environments in which some of previously deleterious mutations may be advantageous. The chaperone Hsp90, whose impairment reveals diverse genetic background dependent phenotypes, was identified as a buffering gene in flies and plants. In yeast, deletion of one endogenous copy of Hsp90 increased population fitness variation under genetic perturbation. However, the actual molecular mechanism of Hsp90-mediated genetic buffering is still unclear. In my studies I seek to elucidate what kinds of genetic loci are buffered by Hsp90 and how this buffering effect is correlated with Hsp90 levels.
I used different strains of Saccharomyces cerevisiae to investigate the degree to which genetic differences among these strains influence phenotypic output when the Hsp90 level is altered. The whole genome expression profile is a quantitative indicator of physiological changes in the cell. Yeast strains with various levels of Hsp90 expression were constructed; the expression profiles of four strains (SK1, M1-2, M2-8 and W303) were compared with the expression profile of a laboratory strain S288C. Genes with the Hsp90-dependent expression difference presumably reflects the consequence of genetic buffering. We identified three kinds of buffering reporter genes. Majority of them were specific to each genetic background, and some of them were overrepresented in certain cellular processes, such as methionine biosynthesis, ergosterol biosynthesis, carbonhydrate transport, and generation of energy. As expected, they are not enriched in the know Hap90 interactors. Therefore, further genetic experiments are required to elucidate how many genetic factors are involved. The relationship between the buffering reporters and Hsp90 will be constructed by combining other genomic information.
A mutation accumulation experiment was designed to study whether genetic buffering is able to constrain mutation distribution and even influences the evolutionary path. Yeast strains with different levels of Hsp90 have accumulated mutations for 2500 generations by passing through single-cell bottleneck. Generally, the evolved cells show fitness declines and aneuploidy patterns, but the current results are not sufficient to answer this question. More discussion on this issue is described in my thesis.
目錄
致謝 2
中文摘要 3
Abstract 5
Introduction 8
A historical review: genetic buffering and canalization 8
Possible mechanisms of genetic robustness 9
The significance of cryptic genetic variation 10
Hsp90 has been regarded as a buffering agent 10
Experimental strategy to understand Hsp90-mediated genetic buffering in yeast 12
Results 14
Genes involved in amino acids biosynthesis show distinct gene expression when Hsp90 is downregulated. 14
Overexpression of Hsp90 is able to increase or diminish gene expression variation in two different yeast strains 15
Ploidy change and increasing production of Hsp90 are common features for mutation accumulated cells 17
Discussion 19
Materials and Methods 26
Yeast strains and medium 26
Yeast protein extraction and Western Blotting 27
RNA extraction 27
Gene expression array 28
DNA content analysis by FACS 29
Growth rate measurement 29
Figures 30
Tables 42
Reference 47
Reference

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