| Summary: | 碩士 === 國立臺灣大學 === 微生物學研究所 === 99 === Giardia lamblia is a flagellated unicellular eukaryotic microorganism that commonly causes diarrheal disease throughout the world. According to the 16s rRNA sequence analysis, G. lamblia has been proposed as an early branching eukaryote. A gene encoding type II DNA topoisomerase was identified in G. lamblia genome. Topoisomerases are enzymes that unwind and wind DNA, in order for genes to be transcribed and to facilitate DNA replication. Myb2 protein is a transcription factor that regulates some genes that were highly expressed during encystation stage. We found that a Myb2 binding site is present in the topoisomerase II gene promoter. The aim of this study is to understand the role of topoisomerase II in Giardia encystation. We found that the endogenous topoisomerase II mRNA levels increases significantly during encystation. The topoisomerase II activity tested by a luciferase reporter system also increased during encystation. The results suggest that topoisomerase II gene may be upregulated during encystation stage and it may have some function in this stage. The sequence of the G. lamblia topoisomerase II is similar to that of the eukaryotic topoisomerase II. EMSA data indicate that topoisomerase II and C-terminal part of topoisomerase II can bind to double stranded DNA, and topoisomerase II and C-terminal part of topoisomerase II have double stranded DNA cleavage ability. ATPase data indicate that topoisomerase II and N-terminal part of topoisomerase II can hydorlyze ATP. Etoposide is an anti-cancer chemotherapy drug. This medication is classified as a topoisomerase II inhibitor. We added etoposide in trophozoite cultures at 400μM concentration and observed cell growth, and found that the cell number decreased significantly 3~24 h after treatment. The results suggest that topoisomerase II may lead to inhibition of G. lamblia cell growth. Addition of 400μM etoposide also resulted a significant decrease of cyst number during 24h encystation, suggesting that topoisomerase II may inhibit G. lamblia differentiation into cysts. Overexpression of topoisomerase II resulted in an increase in the levels of cyst formation and expression of genes for encystation, including cwp1, cwp2 and myb genes, suggesting that topoisomerase II plays an important role in the encystation of G. lamblia.
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