| Summary: | 碩士 === 國立臺灣大學 === 微生物學研究所 === 94 === 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.
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