四膜蟲之計畫性消除染色體內部序列以及小片段染色體之探討

Programmed Elimination of Internal Chromosomal Sequences and Fragmented Chromosomes in Tetrahymena thermophila

林之儀(Chih-Yi Lin)

指導教授 ： 姚孟肇

國立臺灣大學/生命科學院/基因體與系統生物學學位學程/博士(2017年)

https://doi.org/10.6342/NTU201700890

摘要

染色體完整性的維持在基因穩定上是至關重要的，但計畫性去氧核醣核酸重組現象卻可以在許多生物上觀察到。在四膜蟲的的細胞核分化過程中，幾千條序列被切除，並且將五條染色體分割成了幾百條小染色體。雖然核糖核酸已知會標記切除的區域，目前仍未知細胞是如何精準的決定切除邊界。前人研究指出，有些切切除片段的邊界可以由去氧核醣核酸上的順式作用元件決定，進一步推論可能由多種順式作用元件來影響不同群組的內部切除序列。
我們使用基因體定序分析，比較三個四膜蟲野生株之內部切除序列與其在切除邊界上的差異之後發現，內部切除序列在各個野生株內是高度保留的，但同一個內部切除序列在不同野生株上的切除邊界卻有所差異。除此之外，我們還發現有若干種反向重複序列分佈在內部切除序列的兩端。更甚者，同一種反向重複序列到切除邊界的位置在不同內部切除序列上是非常類似的，顯示他們可能作為順式作用元件。最近研究指出，有一個新的反式作用因子 (Lia3) 能夠辨認幾個內部切除序列，包含 M 序列外的順式作用元件 (A5G5)。為了更進一步了解順勢作用元件與其反式作用因子之間的關係，我們比較了野生株以及 Lia3 變異株之內部切除序列的切除邊界變化以尋找由 Lia3 影響之內部切除序列。我們發現有兩個順式作用元件：G-rich 以及 C-rich 的反向重複序列與決定Lia3 影響之內部切除序列的切除邊界非常有關。更有趣的是，這些順式作用元件與切除邊界的距離不受切除邊界差異的影響，進一步顯示順式作用元件是內部切除邊界的決定因子。
接著，我們發現小染色體在染色體切割後有不同的命運。在 326 條體小染色體中，50 條體小染色體在體染色體的發育過程中被選擇性去除了。有趣的是，這些體小染色體都在 6 到 20 次細胞複製之間被去除了。而我們能夠在這些體小染色體上面找到可能具有發育相關功能的基因。我們展示了在纖毛蟲有一種新的計畫性去氧核醣核酸重組模式，這種模式與之前在寄生型線蟲看到的機制類似，並且可能對於發育時期的基因調節上扮演重要的角色。
本篇論文對於計畫性去氧核醣核酸重組以及基因體消除的機制是經由內部序列以及小片段染色體切除的研究上提供了ㄧ個全面性的藍圖。

關鍵字

四膜蟲 ； 計畫性去氧核醣核酸重組 ； 邊界元件 ； 順式邊界元件 ； 染色體切割 ； 端粒 ； 基因體分析

並列摘要

Maintenance of chromosome integrity is crucial for genetic stability. However, programmed DNA rearrangements are known to occur in many organisms. In the ciliate Tetrahymena, thousands of deletion sequences are removed and the five germline chromosomes are fragmented into hundreds of minichromosomes during somatic nuclear differentiation. Although RNA is known to target the region for deletion, it is not clear how the precise boundaries are determined. Previous studies have shown that flanking cis-acting sequences determine the boundaries of some elements and have suggested that there are several cis-acting elements, each affecting a different group of internal elimination sequences (IESs).
Here, we compare IESs and their boundary variations among three wild type Tetrahymena strains by genome sequencing. We show that the IESs are highly conserved among strains even though their boundaries are variable. Furthermore, we have identified several flanking inverted repeats (IRs) at both ends of several IES groups. Localization of these IRs was consistent in groups, suggesting that they could serve as the cis-acting elements. Recently a novel G-quadruplex-binding protein, Lia3, was found to determine the precise boundary of the M element and several IESs flanked by a cis-acting element, A5G5. To further investigate the relationship between cis-acting elements and their binding proteins, we compared the IES boundary variation between wild type strains and Lia3 progeny to identify the group of IES that is affected by Lia3. We found that the G-rich and C-rich IRs were highly correlated with the boundary determination of Lia3-affected IESs. More interestingly, the distances of these IRs to the IES boundaries were consistent even if the boundaries varied among different IES forms, suggesting that the cis-acting elements were the determining factor of IES boundary determinations.
Next, we found that the fates of minichromosomes differ after chromosome breakage. Of the 326 somatic minichromosomes identified using genomic data, 50 were selectively eliminated from the mature somatic genome. Interestingly, many and probably most of these minichromosomes are eliminated during the growth period between 6 and 20 doublings right after conjugation. Genes with potential conjugation-specific functions were found in these minichromosomes. Our findings have revealed a new mode of programmed DNA elimination in ciliates similar to those observed in parasitic nematodes, which could play a role in developmental gene regulation.
The results of this thesis provide a global picture of programmed DNA rearrangement and the mechanism of DNA diminution by internal sequence deletions and minichromosome eliminations in Tetrahymena.

並列關鍵字

Tetrhaymena ； programmed DNA rearrangements ； boundary determination ； cis-acting element ； chromosome breakage ； telomeres ； genomic analysis

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