臺灣博碩士論文加值系統

粒線體與細胞核基因之間的溝通，對於維持細胞正常的生理功能是非常重要的。為了瞭解粒線體與細胞核之間在演化的過程中如何共同適應，先要知道外來的粒線體基因如何影響核基因的表現以及細胞的健康。我使用兩種親源關係相近的酵母菌(Saccharomyces cerevisiae 及 S. paradoxus)來製作細胞質融合但細胞核不融合之「細胞質融合細胞」(cybrids)。觀察酵母菌的生長情形，我發現在正常溫度(28℃)下，生長在YPD (以葡萄糖為唯一碳源之培養液，酵母菌在此培養液中主要行發酵作用來產生能量)及YPGly (以甘油為唯一碳源之培養液，酵母菌在此培養液中只可行呼吸作用來產生能量)的細胞質融合細胞與其細胞核給予者生長情形相似。然而，生長在較高溫(37℃)或較低溫(16℃)時，細胞質融合細胞與核的給予者相比，生長速率就降低許多，這結果顯示了在這些溫度(半限制溫度，37℃或16℃)下，細胞質融合細胞中粒線體的功能可能發生問題。接著，我使用生物晶片(microarray)來檢測細胞質融合細胞中核基因的表現情形。我發現將細胞質融合細胞(ScSpmt)培養在YPGly的培養液中，生長在37℃時，與正常S. cerevisiae細胞相比，會有562細胞核基因發生不正常的調節現象；另外，同樣將細胞質融合細胞(SpScmt)培養在YPGly的培養液中，但是生長在16℃時，與正常的S. paradoxus細胞相比，有1132個基因有不正常調節的現象。有趣的是，我發現細胞核中大部分與粒線體蛋白質轉譯有關的基因，在細胞質融合細胞生長在半限制溫度下表現量會有上升的現象，我發現這些基因表現量的上升可能是為了補償外來粒線體基因所造成的不相容現象。除此之外，細胞質融合細胞(SpScmt)中的細胞色素氧化酶(cytochrome c oxidase)，不管是在正常溫度或是低溫下，都有活性降低的現象，然而在細胞質融合細胞(ScSpmt)中粒線體蛋白的活性卻沒有降低的現象，這結果顯示可能有其他的因素影響細胞質融合細胞的生長。我的研究證明了，即使相近物種的外來粒線體與原來粒線體基因的序列極為相似，仍會影響核基因的表現及細胞的生理作用。

The communication between mitochondrial and nuclear genomes is important for maintaining normal cell metabolism. To understand how these two genomes might have co-adapted during evolution, I examined how foreign mitochondrial DNA influences nuclear gene expression and cell fitness. I used two closely related yeast species, Saccharomyces cerevisiae and S. paradoxus, to construct interspecific cytoductants (cybrids). Results of phenotypic assays showed that the growth rate of cybrids were similar to their nuclear donors on YPD (a medium containing glucose as the sole carbon source, in which yeast cells grow by fermentation) and YPGly (a medium containing glycerol as the sole carbon source, in which yeast cells grow by respiration) plates at the normal growth temperature (28℃). However, I found that the growth rates of cybrids were much slower than those of their parents on glycerol plates at semi-restrictive temperatures (37℃ or 16℃), suggesting that the mitochondrial function in cybrid cells might be compromised at these temperatures. Using gene expression microarrays, I found 562 and 1132 genes have changed their expression levels in S. cerevisiae (ScSpmt) and S. paradoxus (SpScmt) cybrid cells respectively, when the cells were cultured in YPGly medium at semi-restrictive temperatures. Interestingly, most nuclear genes related to mitochondrial protein translation were upregulated in cybrids at semi-restrictive temperatures. It is possible that these genes are upregulated to compensate the deficiency caused by the incompatible foreign mitochondrial genomes. In addition, I observed that the activity of the cytochrome c oxidase, a critical enzyme in the mitochondrial function, was reduced in SpScmt cells but not in ScSpmt. It suggested that there are other factors that can affect the fitness of cybrids. This study demonstrates that a foreign mitochondrial genome can influence not only the expression of nuclear genes but also the cell physiology even though it is from a closely related species and shares high sequence homology.

致謝 ………………………………………………………………………………………………………………….. 3
中文摘要 ………………………………………………………………………………………………………….. 4
Abstract …………………………………………………………………………………………………………... 5
Introduction …………………………………………………………………………………………………….. 7
Results ……………………………………………………………………………………………………………. 11
Discussion ……………………………………………………………………………………………………... 18
Materials and Methods ……………………………………………………………………………………. 22
Figures ………………………………………………………………………………………….……………….. 27
Tables …………………………………………………………………………………………………………….. 43
Reference .………………………………………………………………………………………………………. 47

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