臺灣博碩士論文加值系統

端粒 (telomere) 是真核生物染色體末端一個特化的結構，可用來維持染色體的完整性及穩定性；而端粒的長度則藉由端粒酶 (telomerase) 來調控。在酵母菌Saccharomyces cerevisiae中，端粒酶主要是利用其組成成分中的RNA成員，也就是Tlc1作為模板，並利用特化的反轉錄酶 (reverse transcriptase) Est2p來合成端粒DNA。目前已發現數種蛋白質可藉由與Tlc1作用而影響端粒酶的活性，例如Ku70/80p以及Est1p等。這些蛋白在Tlc1上的結合位置已經被定義出來，且這些結合位置對於Tlc1的功能而言是不可或缺的，但是若只有目前這幾個蛋白與Tlc1之間的作用，卻不足以維持完整的端粒酶活性。因此，為了尋找是否有其他能藉由與Tlc1作用，而調控端粒酶活性的蛋白存在，我們在酵母菌中建立了一個三雜交系統 (yeast three-hybrid system)，來尋找可和Tlc1結合的蛋白。我們將Tlc1與RRE (Rev responsive element) 融合形成一個RRE-Tlc1雜合RNA (hybrid RNA)，此雜合RNA可藉由與Rev蛋白作用，而被與Rev融合在一起的Gal4 DNA binding domain帶到報告基因(reporter genes)上游的UAS序列上。而帶有酵母菌基因體DNA片段的DNA基因庫(DNA library)，則與Gal4 activation domain融合，以便於篩選。利用此三雜交系統，我們已經成功篩選出兩個可與Tlc1作用的蛋白，然而這兩個蛋白在調控端粒酶活性中扮演何種角色，則需要再做進一步分析與探討。

Telomere is a specialized structure of chromosome end for maintaining the chromosome integrity and stability in eukaryotes. Telomere elongation is dependent on a ribonuclearprotein, telomerase. In Saccharomyces cerevisiae, telomerase utilizes its RNA component, Tlc1, as the template to copy telomeric DNA. The synthesis of telomeric DNA sequences is achieved by a specialized reverse transcriptase Est2p. Several protein factors were also found to interact with Tlc1. For example, Ku70/80p heterodimer and Est1p were shown to bind to Tlc1 RNA. Together these proteins participate in regulating telomerase activity. The Tlc1 RNA binding regions of these proteins were mapped. Although it was shown that these binding regions are required for telomerase activity, they are not sufficient for proper telomerase function. To search for additional Tlc1-interacting proteins that might affect telomerase function, we established a three-hybrid screening system in yeast. This system enables the identification of proteins that interact with Tlc1 RNA. Here we fused Tlc1 with RRE (Rev responsive element) to generate a construct that expressed a Tlc1-RRE hybrid RNA. The hybrid RNA could be recruited to the UAS of reporter genes by a fusion protein that the Gal4 DNA binding domain was fused to a RRE-binding protein, RevM10. DNA library carry the yeast genomic DNA fragments fused with the activation domain of Gal4 were used for screenings. Using this established system, we have identified two novel genes that interacted with Tlc1 RNA. The telomere functions of these genes will be analyzed and discussed.

論文電子檔著作權授權書 ………………………………………… i
論文審定同意書 …………………………………………………… ii
誌謝 ………………………………………………………………… iii
中文摘要 …………………………………………………………… iv
英文摘要 …………………………………………………………… v
目錄 ………………………………………………………………… vi
圖目錄 ……………………………………………………………… vii
緒論 ………………………………………………………………… 1
材料與方法 ………………………………………………………… 7
結果 ………………………………………………………………… 25
討論 ………………………………………………………………… 30
參考文獻 …………………………………………………………… 33

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