gPax2, a Pax homologue in Giardia lamblia, involved in transcriptional regulation of cyst wall protein 1 and 2 genes
| Autor: | Shen-Fung Chuang, 莊勝帆 |
|---|---|
| Rok vydání: | 2009 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 97 Pax protein family is a large group of transcription factors in vertebrates. They are mainly involved in embryogenesis and organogenesis. To date, the pax gene has still not been found in protozoa. We have found two Pax-like open reading frames, gPax1 and gPax2, in Giardia lamblia data base. Unlike Pax proteins from vertebrates which have a paired DNA-binding domain near the N terminus, the paired domains of gPax1 and gPax2 were located near the C terminus. We found that the mRNA expression levels of endogenous gpax2 gene during 24 hours encystation were lower than that during vegetative growth in G. lamblia. We transfected a construct that expressed HA-tagged gpax2 into wild type G. lamblia. Western blot showed that HA-tagged gPax2 was expressed at similar levels during vegetative growth and during 24 hr encystation stage in G. lamblia. Immunofluorescence assay showed that the HA-tagged gPax2 was located in nuclei during vegetative growth and 24 hr encystation in G. lamblia. By electrophoretic mobility shift assays, we found that gPax2 specifically bound to the promoters of cyst wall protein 1 (cwp1) and cwp2 which are important genes during stages-changing in G. lamblia. Cwp1 and Cwp2 are two components of cyst wall. The expression of the cwp1and cwp2 mRNA was induced during encystation in G. lamblia. We made a mutant, gPax2del, which lacks the C-terminal paired domain. We found that gPax2del could not bind to the cwp1 and cwp2 promoters. gPax2del was expressed in some vesicles in cytosol, and gPax2del proteins were hardly detected in nuclei. We found that the levels of the cwp1 and cwp2 mRNA and Cwp1 protein in the gPax2 overexpressing cell line increased significantly relative to the levels in the vector control cell line. In addition, we found that the levels of the cwp1 and cwp2 mRNA and Cwp1 protein in the gPax2del overexpressing cell line decreased significantly relative to the levels in the gPax2 overexpressing cell line. We suggest that gPax2 might be involved in the transcriptional regulation of the cwp1 and cwp2 genes, and the paired domain would contribute partially to this regulation. We found two stretches of basic amino acids that may be nuclear localization signals (NLS)-like sequences in the paired domain of gPax2. We made two mutants, gPax2m1 and gPax2m2, by mutating the positive charge amino acids to neutral amino acids. We found that gPax2m1 and gPax2m2 were located in cytosol, and around the sucking disk, but gPax2m1 and gPax2m2 were hardly detected in nuclei. This suggests that these two stretches of basic amino acids in the paired domain could be NLSs. We found that the levels of the cwp1 and cwp2 mRNA and Cwp1 protein in the gPax2m1 overexpressing cell line decreased significantly relative to the levels in the gPax2 overexpressing cell line. The levels of the cwp1 and cwp2 mRNA and Cwp1 protein in the gPax2m2 overexpressing cell line increased significantly relative to the levels in the gPax2 overexpressing cell line. We suggest that two NLSs may be present in the paired domain of gPax2 and they could influence the cwp1 and cwp2 gene expression. We suggest that gPax2 can up-regulate the cwp1 and cwp2 gene expression and the paired domain of gPax2 might be involved in this regulation. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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