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The effects of puromycin and G418 on cwp gene expression and functional characterization of the Myb transcription factor in Giardia lamblia
No full text梨形鞭毛蟲為寄生在人體小腸中之致病性原蟲。目前研究梨形鞭毛蟲基因功能常使用穩定轉染系統，此系統需使用G418和嘌呤黴素作為篩選蟲株的藥劑，但是目前這些藥劑對梨形鞭毛蟲基因表現的影響並不清楚。此研究中，我們發現G418、嘌呤黴素或穩定轉染系統本身能增加cwp1、cwp2和gmyb2基因表現約1.3-6倍。我進一步利用nuclear run on assay發現此cwp1、cwp2和gmyb2基因RNA增加的現象，有一部分來自轉錄起始速率增加。當梨形鞭毛蟲處於囊體化環境下，G418或嘌呤黴素卻少許抑制或不影響cwp1和cwp2的表現。此外，穩定轉染細胞株會促進囊體的形成，約1.6∼2.1倍，可能是由於這些細胞株之cwp基因表現較高所致。但是G418或嘌呤黴素卻會降低囊體的形成，可能是由於這些藥物抑制梨形鞭毛蟲的生長速度。由此研究結果能了解穩定轉染系統會增加梨形鞭毛蟲囊體化基因表現及囊體形成。
由於此蟲需經過囊體化形成囊體才能存活於外界，所以囊體化為此蟲重要的調控機制。但是目前對囊體化相關基因調控並不清楚，我發現當大量表現滋養體時期中的gmyb2基因時，能使內生性cwp1和cwp2基因表現量上升並且使細胞株囊體形成效率變好。利用基因抑制法降解gmyb2 RNA能使內生性cwp1和cwp2基因表現量下降，並且使細胞株囊體形成效率變差，這些結果顯示gMyb2可能是重要的轉錄活化子，可以促使cwp基因的表現和囊體的形成。我更進一步觀察在cwp3基因coding region的gMyb2結合序列的角色，結果發現改變gMyb2結合序列中的鹼基，並不影響cwp3基因表現。若改變cwp3基因的coding region中之gMyb2結合序列所轉譯出的胺基酸序列，會造成cwp3基因表現下降。另外，我發現大量表現cwp3基因時可增加囊體形成。我的研究結果提供了梨形鞭毛蟲生活史的轉錄調控更深入的了解。Giardia lamblia is an important human intestinal parasite causing giardiasis. Two stable DNA transfection systems, under the selection of neomycin and puromycin have been established. Very little is known about how these drug selection systems may influence gene expression in Giardia. Therefore, I tested the hypothesis that these drug selection systems themselves might have effects on general expression of other genes. Northern blot analysis showed a 1.3 to 6 fold coordinate increase in the encystation-induced cwp1, cwp2, and gmyb2 gene transcripts in vegetative cells treated with G418 or puromycin, or cells stably transfected with vectors containing the drug selectable genes. Nuclear run on assays showed that the effects of G418, puromycin, or stable transfection systems, at least in part, were due to an increased rate of transcription. However, during encystation, the drug treatment or stable transfection did not influence the cwp1, cwp2, and gmyb2 mRNA levels. I further found that the levels of cyst formation in the cells stably transfected with vectors containing the drug selectable genes increased to 1.6~2.1 fold of that of the non-transfected wild type cells. This could be due to the higher expression of the cwp genes in the stable transfectants. However, wild type cells treated with G418 or puromycin demonstrated a decreased cyst count, compared with untreated wild type cells, indicating that these procedures reduce the encystation efficiency. This could be due to the lower growth rate in the drug treated cells. My results indicate that these stable transfection systems can increase expression levels of encystation specific genes and cyst formation in G. lamblia.
G. lamblia survives outside of the host by differentiation of trophozoites into infectious cysts. Transcriptional regulation is key for encystation-specific gene expression, but the mechanisms are still not clear. In my thesis, I found that overexpression of the gmyb2 gene resulted in an increase of both endogenous cwp1 and cwp2 gene expression and cyst formation. In addition, knockdown of the gmyb2 gene resulted in a decrease of the endogenous cwp1 and cwp2 gene expression and cyst formation. These results indicate that gMyb2 may be an important transactivator for up-regulating cwp gene expression and cyst formation in G. lamblia. I further investigated the role of the gMyb2 binding in the cwp3 gene coding region. Mutation of the gMyb2 binding site but not change any amino acid sequence of the cwp3 coding region resulted in no change in the cwp3 mRNA and protein levels. However, mutation of the gMyb2 binding site and change of one amino acid sequence resulted in a decrease in the cwp3 mRNA and protein levels. Furthermore, I also found that overexpression of the cwp3 gene resulted in an increase of cyst formation. My studies provide new insights into transcriptional regulation of the life cycle of G. lamblia.前言………………………………………………..……………….........1
材料與方法
一、梨形鞭毛蟲細胞株的培養………………………………………6
二、培養梨形鞭毛蟲所使用之嘌呤黴素、G418和四環黴素的劑量與培養方式………………………………………………………6
三、梨形鞭毛蟲囊體化培養方式……………………………………6
四、轉殖質體的建構與萃取…………………………………………6
五、梨形鞭毛蟲轉染與選殖…………………………………………10
六、囊體和囊體化滋養體計數法……..……………………..………11
七、北方墨點法……………………….……………………….……..11
八、Nuclear run on分析法.……………………….…………….…....12
九、西方墨點法……………………..………………………………..13
結果
一、分析嘌呤黴素、G418和四環黴素對滋養體時期之囊體化相關基因表現之影響……………………..…………………………16
二、分析嘌呤黴素、G418和四環黴素對梨形鞭毛蟲細胞株囊體形
成情形之影響…………………….………………………….…17
三、分析嘌呤黴素、G418和四環黴素對囊體化時期之囊體化相關
基因表現之影響……………………...…………….…………...18
四、分析gMyb2蛋白的功能.………………………….……………19
五、分析gMyb2蛋白對梨形鞭毛蟲細胞株囊體形成情形之影響..20
六、利用基因抑制法分析gMyb2對梨形鞭毛蟲基因之影響…….21
七、分析pmybas細胞株囊體形成情形…………….………………22
八、cwp3基因coding region之gMyb2結合序列之分析……………22
九、CWP3蛋白對囊體形成之影響…………..……………………..23
討論………………………………………………………………..….25
附圖…………………………………………………….…………..…28
圖表…………………………………..…….……………...……….…31附表….……………………………….…………………………….....41
參考文獻………………………………………………………….…4
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