黑腹果蠅端粒TART反轉子ORF2p內切酶之表現、純化及活性測試

Expression,purification and activity of the ORF2p endonuclease from TART retrotransposon of Drosophila telomere

曾奕之(Yi-Chih Tzeng)

指導教授 ： 陳銘凱

淡江大學/理學院/生命科學研究所碩士班/碩士(2008年)

https://doi.org/10.6846/TKU.2008.00702

摘要

黑腹果蠅（Drosophila melanogaster）易於培養和繁殖。在基因研究方面，果蠅是最常見的研究對象，它有四對染色體，且可以顯示很多變異，所以我們經常利用它作基礎的研究。端粒的長短與生物老化的過程息息相關，希望能藉由黑腹果蠅端粒的研究模型，了解端粒之演化基礎及物種老化的過程。
黑腹果蠅其端粒是由三種的non-LTR的retrotransposons組成，其分別為HeT-A、TART和TAHRE的重複序列，而其中TART可以轉譯出兩種蛋白質，ORF1p和ORF2p。TART ORF1p已證明是當共表現HeT-A ORF1p時，用來移動HeT-A ORF1p到telomere-associated structures上的蛋白質。而ORF2p則可能具有endonuclease（EN）和reverse transcriptase（RT）的活性。
本實驗是研究TART-EN的部分，目前已將endonuclease、點突變蛋白和二種的截短蛋白大量的表現在最適合的E.coli宿主上，並且能夠在具有活性的前提之下把endonuclease純化出來並做有效的保存。已確定此種endonuclease確實具有活性，可以將supercoiled form DNA的受質切成open circular form DNA。在活性確認之後，便可進而研究此endonuclease之DNA切位專一性以及辨認序列，以瞭解TART之作用方式。

關鍵字

黑腹果蠅 ； 內切酶

並列摘要

The Drosophila melanogaster is easy to culture and propagation. In gene studies, the Drosophila melanogaster is the most common experimental animal. It has four pairs of chromosome, and have much genetic variation, so it is often used in basic research. The length of the telomere is closely correlated with aging process. We hope that by studying the Drosophila melanogaster's telomere we can understand the evolution of telomere and the aging process in general.
Drosophila melanogaster telomere is composed of three non-LTR retrotransposons, HeT-A, TART and TAHRE, which are repeated sequences, and TART can translate two kinds of proteins, ORF1p and ORF2p. TART ORF1p has already proved its function in shifting the telomere-associated structures only when coexpressed with HeT-A ORF1p. And ORF2p might have endonuclease (EN) and reverse transcriptase (RT) activity.
In this study, we investigate TART-EN expression and purification. We have already over-expressed Endop, point-mutant Endop, and two truncated mutant Endop in suitable E.coli. We Confirmed Endop have endonuclease activity by cutting supercoiled form DNA into open circular form DNA. We can also keep endonuclease activity of purified Endop effectively by storing it in 30％ glycerol and -20℃. After the enzyme activity affirmation, we can study the cutting hot spots of endonuclease and identify their sequences further in order to understand the function of TART.

並列關鍵字

Drosophila melanogaster ； TART ； endonuclease

參考文獻

Richard M Cawthon, Ken R Smith, Elizabeth O'Brien, Anna Sivatchenko and Richard A Kerber (2003) Association between telomere length in blood and mortality in people aged 60 years or older. The Lancet, Volume 361, Issue 9355, 1 February 2003, Pages 393-395.

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Rashkova, S., Karam, S.E. and Pardue, M.L. (2002b) Element-specific localization of Drosophila retrotransposon Gag proteins occurs in both nucleus and cytoplasm. Proc Natl Acad Sci U S A, 99, 3621-3626.

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