Neomycin and puromycin affect gene expression in Giardia lamblia stable transfection

Su, Li; Lee, Gilbert; Yu, Haomiao; Chen, Yi Hsiu; Sun, Chin-Hung

doi:10.1016/j.molbiopara.2007.07.015

Cited by 40 publications

(48 citation statements)

References 53 publications

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“…We obtained consistent cyst count data for vegetative G. lamblia cultures during growth to stationary phase (ϳ4800 cysts/ml for 5Ј⌬5N-Pac cell line) (64). In this study, we found that the cyst number in the Cdk2 overexpressing cell line increased by ϳ2.0-fold (p Ͻ 0.05) relative to the control cell line, which expresses only the puromycin selection marker (5Ј⌬5N-Pac) (Fig.…”

Section: Overexpression Of Cdk2 Induced Expression Of Cwp1 Cwp2supporting

confidence: 76%

“…Briefly, trophozoites that were grown to late log phase in growth medium were harvested and encysted for 24 h in TYI-S-33 medium containing 12.5 mg/ml of bovine bile at pH 7.8 at a beginning density of 5 ϫ 10 5 cells/ml. Cyst Count-Cyst count was performed on the stationary phase cultures (ϳ2 ϫ 10 6 cells/ml) during vegetative growth as previously described (64) and the data shown in Fig. 3C.…”

Section: Methodsmentioning

confidence: 99%

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Regulation of a Myb Transcription Factor by Cyclin-dependent Kinase 2 in Giardia lamblia

Cho

Huang

et al. 2012

Journal of Biological Chemistry

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Background: Giardia lamblia parasitizes the human small intestine to cause diseases. It differentiates into infectious cysts by intestinal stimulation. Results: Giardial Cdk2 is associated with up-regulation of cyst wall protein genes. Conclusion: Cdk2 may phosphorylate a Myb transcription factor, leading to activation of cyst wall protein genes during encystation. Significance: The results may help us to develop new drugs to inhibit cyst formation by disrupting the Cdk2 pathway.

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Section: Overexpression Of Cdk2 Induced Expression Of Cwp1 Cwp2supporting

confidence: 76%

Section: Methodsmentioning

confidence: 99%

Regulation of a Myb Transcription Factor by Cyclin-dependent Kinase 2 in Giardia lamblia

Cho

Huang

et al. 2012

Journal of Biological Chemistry

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“…The induction levels of CWP mRNA expression reported in the literature range from 100-to 300-fold with low background expression in trophozoites (11,21,27,40). Considering that there is a spontaneous rate of encystation of up to a few percent in standard trophozoite cultures (38), it is more likely that CWP expression is completely silenced at that developmental stage. Peak mRNA levels of the most abundant proteins exported to the cyst wall (CWP1-3) in induced parasites are detected at ϳ7 h p.i.…”

Section: Resultsmentioning

confidence: 99%

The Transcriptional Response to Encystation Stimuli in Giardia lamblia Is Restricted to a Small Set of Genes

Morf

Spycher

Rehrauer³

et al. 2010

Eukaryot Cell

111

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The protozoan parasite Giardia lamblia undergoes stage differentiation in the small intestine of the host to an environmentally resistant and infectious cyst. Encystation involves the secretion of an extracellular matrix comprised of cyst wall proteins (CWPs) and a ␤(1-3)-GalNAc homopolymer. Upon the induction of encystation, genes coding for CWPs are switched on, and mRNAs coding for a Myb transcription factor and enzymes involved in cyst wall glycan synthesis are upregulated. Encystation in vitro is triggered by several protocols, which call for changes in bile concentrations or availability of lipids, and elevated pH. However, the conditions for induction are not standardized and we predicted significant protocol-specific side effects. This makes reliable identification of encystation factors difficult. Here, we exploited the possibility of inducing encystation with two different protocols, which we show to be equally effective, for a comparative mRNA profile analysis. The standard encystation protocol induced a bipartite transcriptional response with surprisingly minor involvement of stress genes. A comparative analysis revealed a core set of only 18 encystation genes and showed that a majority of genes was indeed upregulated as a side effect of inducing conditions. We also established a Myb binding sequence as a signature motif in encystation promoters, suggesting coordinated regulation of these factors.The differentiation of Giardia lamblia cells to cysts is a key step in the simple life cycle of this ubiquitous intestinal parasite. Induced trophozoites, the flagellated, motile, and attachment-competent stage, exit the cell cycle at the G 2 -M transition (1) and begin to synthesize the components of an extracellular matrix, the cyst wall (CW). Synthesis and export of the cyst wall proteins (CWPs) to specialized organelles, termed encystation-specific vesicles (ESVs), is completed between 8 and 10 h postinduction (p.i.). After proteolytic cleavage of the CWP2 C terminus, the CWPs are sorted into two fractions which are deposited sequentially and polymerize on the surface of the cyst (18). The first layer of the CW, which eventually completely encloses the parasite (19,24), is secreted in the last minutes of differentiation simultaneously with nuclear division and morphological transformation of the cell (cyst formation). In vitro, the entire encystation process typically takes 20 to 24 h in our hands and can be induced by modifying medium components and/or the concentrations of bile, fatty acids (cholesterol), or lactic acid (3,10,15,22). Because the condition(s) which induce encystation in vivo are not known, all factors and components mentioned above are implicated since their concentrations vary in the small intestine, as does the pH, which increases from ϳ6 in the duodenum to 7.5 to 8.0 in the distal ileum. Although all available data indicate that differentiation is induced by one or several environmental signals, the mechanism(s) for reception and transduction have not been characterized. Upregulation...

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“…The cyst count was performed on the stationary phase cultures (∼2×10 6 cells/ml) during vegetative growth as previously described [51]. Cells were subcultured in growth medium with suitable selection drugs at an initial density of 1×10 6 cells/ml.…”

Section: Methodsmentioning

confidence: 99%

Functional Redundancy of Two Pax-Like Proteins in Transcriptional Activation of Cyst Wall Protein Genes in Giardia lamblia

Chuang

Cho

et al. 2012

PLoS ONE

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The protozoan Giardia lamblia differentiates from a pathogenic trophozoite into an infectious cyst to survive outside of the host. During encystation, genes encoding cyst wall proteins (CWPs) are coordinately induced. Pax family transcription factors are involved in a variety of developmental processes in animals. Nine Pax proteins have been found to play an important role in tissue and organ development in humans. To understand the progression from primitive to more complex eukaryotic cells, we tried to identify putative pax genes in the G. lamblia genome and found two genes, pax1 and pax2, with limited similarity. We found that Pax1 may transactivate the encystation-induced cwp genes and interact with AT-rich initiatior elements that are essential for promoter activity and transcription start site selection. In this study, we further characterized Pax2 and found that, like Pax1, Pax2 was present in Giardia nuclei and it may specifically bind to the AT-rich initiator elements of the encystation-induced cwp1-3 and myb2 genes. Interestingly, overexpression of Pax2 increased the cwp1-3 and myb2 gene expression and cyst formation. Deletion of the C-terminal paired domain or mutation of the basic amino acids of the paired domain resulted in a decrease of nuclear localization, DNA-binding activity, and transactivation activity of Pax2. These results are similar to those found in the previous Pax1 study. In addition, the profiles of gene expression in the Pax2 and Pax1 overexpressing cells significantly overlap in the same direction and ERK1 associated complexes may phosphorylate Pax2 and Pax1, suggesting that Pax2 and Pax1 may be downstream components of a MAPK/ERK1 signaling pathway. Our results reveal functional redundancy between Pax2 and Pax1 in up-regulation of the key encystation-induced genes. These results illustrate functional redundancy of a gene family can occur in order to increase maintenance of important gene function in the protozoan organism G. lamblia.

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Neomycin and puromycin affect gene expression in Giardia lamblia stable transfection

Cited by 40 publications

References 53 publications

Regulation of a Myb Transcription Factor by Cyclin-dependent Kinase 2 in Giardia lamblia

Regulation of a Myb Transcription Factor by Cyclin-dependent Kinase 2 in Giardia lamblia

The Transcriptional Response to Encystation Stimuli in Giardia lamblia Is Restricted to a Small Set of Genes

Functional Redundancy of Two Pax-Like Proteins in Transcriptional Activation of Cyst Wall Protein Genes in Giardia lamblia

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