酵母菌基因體的演化分析研究

Abstract

Saccharomyces cerevisiae經由古老的全基因體複製（whole-genome duplication，WGD）產生的重複基因（duplicate genes）中，有許多基因對之間表現出了較預期低許多的同義分歧（synonymous divergence，KS），有些基因對之間的序列相似性比該基因和S. bayanus的同源基因（orthologue）之間的相似性更高，或者是和Kluyveromyces waltii（在WGD發生之前分化的物種）的同源基因相比，擁有較慢的演化速度。這樣的減速演化（decelerated evolution）過去被歸因於重複基因之間的基因轉換（gene conversion）。在這篇論文的第一部份，我探討了四個物種中約三百個WGD基因對，以及這些基因對在非WGD物種中的同源基因，並因此發現了密碼子使用偏移（codon usage bias）以及蛋白質序列的保守性是造成重複基因對的減速演化兩個重要的原因。基因轉換只有在巨大的密碼子使用偏移或是非常保守的蛋白質序列存在的情況下，才能有效地對減速演化造成影響。我更進一步發現，突變型態的改變，或是tRNA編碼基因拷貝數目（tDNA copy number）的改變，會造成密碼子使用偏移的改變，也因此導致K. waltii及S. cerevisiae之間同義距離（KS distance）的增加。很有趣地，有些蛋白質在WGD物種輻射狀種化之前表現出很快的演化速率，然而，在輻射狀種化之後他們的演化速率卻降得很低，甚至不再有變化。這代表功能上的保守性對於重複基因對的減速演化也有很大的影響。 接下來，我利用功能性基因體及蛋白質結構資料，探討蛋白質複雜性（蛋白質次單元種類的數目，protein complexity）對基因的可移除性（dispensability）及可複製性（duplicability）的影響。結果發現，基因可複製性在異複合體（由兩種以上的次單元所構成的複合體，hetero-complexes）及同複合體（單體或是單一種次單元所構成的複合體，homo-complexes）之間存在明顯的差異。然而，基因的可移除性則是隨著蛋白質複合體次單元種類數目的增加而逐漸降低。這代表劑量平衡假說（dosage balance hypothesis）雖然能夠解釋蛋白質複合體的基因可複製性，卻無法完美的解釋不同的異複合體之間基因可移除性的差異。可能的情況是當一個異複合體次單元基因被剔除的時候，整個複合體的功能都會受到影響。因此這個基因被剔除造成的適性（fitness）影響會隨著蛋白質複雜性的升高而增加。此外我發現具有多功能區（multi-domain）的多肽基因和只具有單一功能區的多肽基因相比，有較低的可移除性和較高的可複製性。經由WGD產生的重複基因（不含核糖體次單元基因）普遍的比其他的重複基因擁有較高的可移除性。而屬於同一個複合體的次單元通常傾向有類似的表現量和類似的剔除適性影響。最後，我估計重複基因對於基因突變頑抗性（genetic robustness against null mutation）的貢獻大約是9%，比前人估計的要小許多。對酵母菌的基因可移除性來講，蛋白質的複雜性應該比重複基因的影響來的重大許多。 最後我所探討的是蛋白質演化速率。最近的研究指出，酵母菌中蛋白質的演化速率唯一的主要決定因素在於轉譯效率的選擇力（translational selection）上，這可以由mRNA和蛋白質的表現量以及密碼子適應值（codon adaptation index）來表示。本研究則說明蛋白質的結構其實也有舉足輕重的影響。為了要維持蛋白質的結構穩定，包埋在蛋白質內部或是位於蛋白質交互作用面的殘基（residue）通常比暴露在蛋白質表面接觸溶劑的殘基面對更強的演化拘束力。經由淨相關（partial correlation）分析發現，蛋白質中暴露殘基的百分比（Pexposed）可以解釋的演化速率變異量，可達到轉譯效率的選擇力所能解釋的一半以上。這個結果和功能性密度（functional density）假說是一致的，也就是說，蛋白質若擁有較多殘基與特定功能相關（如穩定蛋白質結構或是蛋白交互作用），會因此而傾向擁有較慢的演化速率。

Many Saccharomyces cerevisiae duplicate genes that were derived from an ancient whole-genome duplication (WGD) unexpectedly show a small synonymous divergence (KS), a higher sequence similarity to each other than to orthologues in S. bayanus, or slow evolution compared to the orthologue in Kluyveromyces waltii, a non-WGD species. This decelerated evolution was attributed to gene conversion between duplicates. Using ~300 WGD gene pairs in four species and their orthologues in non-WGD species, the first part of my thesis shows that codon usage bias and protein sequence conservation are two important causes for decelerated evolution of duplicate genes, whereas gene conversion is effective only in the presence of strong codon usage bias or protein sequence conservation. Further, I found that change in mutation pattern or in tDNA copy number changed codon usage bias and increased the KS distance between K. waltii and S. cerevisiae. Intriguingly, some proteins showed fast evolution before the radiation of WGD species but little or no sequence divergence between orthologues and paralogues thereafter, indicating that functional conservation after the radiation may also be responsible for decelerated evolution in duplicates. In the second part, I studied the effects of protein complexity (here defined as the number of subunit types in a protein) on gene dispensability and gene duplicability using functional genomic and protein structural data. I found that the major distinction for gene duplicability in protein complexity is between hetero-complexes, each of which includes at least two different types of subunits (polypeptides), and homo-complexes, which include monomers and complexes that consist of only subunits of one polypeptide type. However, gene dispensability decreases only gradually as the number of subunit types in a protein complex increases. These observations suggest that the dosage balance hypothesis can explain gene duplicability of complex proteins well, but cannot completely explain the difference in dispensabilities between hetero-complex subunits. It is likely that knocking out a gene coding for a hetero-complex subunit would disrupt the function of the whole complex, so that the deletion effect on fitness would increase with protein complexity. I also found that multi-domain polypeptide genes are less dispensable but more duplicable than single domain polypeptide genes. Duplicate genes derived from the whole genome duplication event in yeast are more dispensable (except for ribosomal protein genes) than other duplicate genes. Further, I found that subunits of the same protein complex tend to have similar expression levels and similar effects of gene deletion on fitness. Finally, I estimated that in yeast the contribution of duplicate genes to genetic robustness against null mutation is ~ 9%, smaller than previously estimated. In yeast, protein complexity may serve as a better indicator of gene dispensability than do duplicate genes. The last part is a study related to protein evolutionary rate. Recently, translational selection, including mRNA expression, protein abundance, and codon adaptation index, has been suggested as the single dominant determinant of protein evolutionary rate in yeast. This study shows that protein structure is an important determinant as well. Buried residues, which are responsible for maintaining protein structure or located on a stable interaction surface, are under stronger constraints than solvent-exposed residues. Partial correlation analysis shows that the variance of evolutionary rate explained by the proportion of exposed residues (Pexposed) can reach more than half of that explained by translational selection. This result suggests that proteins with many residues involved in specific functions (e.g. maintaining structure or protein interaction) may evolve more slowly, which is consistent with the “functional density” hypothesis.