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

• Main Authors: Yi-Chih Tzeng, 曾奕之
• Other Authors: Ming-Kai Chen
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/29544772337613747656

碩士 === 淡江大學 === 生命科學研究所碩士班 === 96 === 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.