鑑定梨形鞭毛蟲的Pax同源蛋白質對於cyst wall protein 1 和 2基因的轉錄調控之影響

Abstract

Pax proteins with paired domains have been identified in Drosophila, nematodes, and vertebrates. Pax proteins have been demonstrated as specialized transcription factors involved in cell proliferation and development. To date, pax-like gene has not been found in plant, protozoa and fungi. We found one Pax-like open reading frame from Giardia lamblia genome data base and we named it gPax1. gPax1 has a paired domain near its C terminus as predicted by SMART program. During vegetative growth and encystation stage, endogenous gPax1 gene was expressed at similar levels of mRNA. To understand the function of gPax1, we transfected a construct which expressed AU1-tagged gpax1 gene into G. lamblia. Inmunofluorescence assay and Western blot analysis showed that the AU1-tagged gPax1 was localized to neclei and expressed at similar protein levels during vegetative growth and encystation stages. Using Electrophoretic Mobility Shift assays (EMSA), we found that gPax1 can bind to the promoter of cyst wall protein 1 (cwp1) and cwp2 genes, suggesting that gPax1 could be a transcription factor and involved in transcriptional regulation of the cwp1 and cwp2 genes. Mutation analysis revealed that gPax1 prefered to bind to AT-rich sequence. Overexpression of gPax1 resulted in a significant increase of levels of cwp1 and cwp2 mRNA and of the Cwp1 protein. A gPax1 mutant, gPax1del, that lacks the paired domain was analyzed. We found that gPax1del lost the binding ability to the promoter of the cwp1 and cwp2 genes. gPax1del was localized to both nuclei and cytoplasm. We also found that the levels of the cwp1 and cwp2 mRNA and Cwp1 protein in the gPax1del overexpressing cell line decreased significantly relative to the levels in the gPax1 overexpressing cell line, suggesting that the paired domain of gPax1 could be very important to the regulation ability of gPax1. Two stretches of basic amino acids were found in the paired domain of gPax1. We tried to understand whether these two regions are nuclear localization signal (NLS). When we mutated the basic amino acids in these two regions to neural amino acids (gPax1m1 and gPax1m2 mutants), the binding ability to the promoter of the cwp1 and cwp2 genes was lost. gPax1m1 was localized to nuclei. We found that the levels of the cwp1 and cwp2 mRNA and Cwp1 protein in the gPax1m1 overexpressing cell line decreased relative to the levels in the gPax1 overexpressing cell line. gPax1m2 was localized to some vesicles in cytoplasm. The levels of the cwp1 and cwp2 mRNA and Cwp1 protein in the gPax1m2 overexpressing cell line decreased significantly relative to the levels in the gPax1 overexpressing cell line. One stretch of basic amino acids in the paired domain could be NLS as shown by the results from the gPax1m2 mutant. Mutation of this potential NLS resulted in a decrease of nuclear localization and the regulation ability of gPax1. Mutation of another stretch of basic amino acids in the paired domain did not change the localization of gPax1 to the nuclei, but this mutation still decreased the DNA binding and transactivation ability of gPax1 (gPax1m1). We suggested that gPax1 may be an important transcriptional activator in regulation of the cwp1 and cwp2 genes, and the paired domain may contribute to the function of gPax1.