title: Influence of 5' sequences on expression of the Tet repressor in Giardia lamblia.
abstract: Gene expression is poorly understood in Giardia lamblia. Previously we utilized the Escherichia coli tetracycline regulatory elements to develop a giardial-inducible gene expression system. In this study, we tested the hypothesis that regions flanking the tet repressor (tet R) may influence its expression and affect inducibility of the regulatory system. We found that addition of a 6-His tag or nuclear localization signal (NLS) at the N- but not C-terminus of tet R, increased the induction ratios >100-fold. A non-specific sequence also increased the induction ratio. Fusing NLS at the N-terminus, also led to exclusively nuclear tet R localization. Changing the promoter from gdh or alpha-giardin to alpha2-tubulin increased the induction ratio slightly. Tet R expression at both RNA and protein levels correlated with repression efficiency, indicating that coding sequences of the 6-His tag or NLS may contribute to transcriptional activation of the exotic tet R gene in Giardia. In addition, we found that the tet R system mediated gene repression and induction during encystation. Previous studies used an artificial reporter gene. In this study, we were able to induce overexpression of epitope-tagged cyst wall protein 1 (CWP1) in vegetatively growing Giardia trophozoites. Moreover, we could repress or induce expression of exogenous CWP1 in encysting cells. Taken together, our data suggest that expression of tet R in Giardia is complex and can be strongly influenced by additional sequences, especially at its N-terminus. This system provides insights into expression of an alien gene and can be exploited to regulate gene expression and study important functions in G. lamblia.
authors.affiliation: Department of Parasitology, College of Medicine, National Taiwan University, Taipei, 100 Taiwan, ROC. chsun@ha.mc.ntu.edu.tw
authors.forename: Chin-Hung
authors.initials: CH
authors.lastname: Sun
authors.forename: Li-Hsin
authors.initials: LH
authors.lastname: Su
authors.forename: Frances D
authors.initials: FD
authors.lastname: Gillin
mesh_terms: D024506:5' Flanking Region; D000818:Animals; D005786:Gene Expression Regulation; D016829:Giardia lamblia / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D006639:Histidine; D019913:Nuclear Localization Signals; D011401:Promoter Regions, Genetic; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: C094333:CWP1 protein, Giardia lamblia; D019913:Nuclear Localization Signals; D015800:Protozoan Proteins; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D006639:Histidine