Functionality of the GAL4/UAS system in Tribolium requires the use of endogenous core promoters

<p>Abstract</p> <p>Background</p> <p>The red flour beetle <it>Tribolium castaneum </it>has developed into an insect model system second only to <it>Drosophila</it>. Moreover, as a coleopteran it represents the most species-rich metazoan taxon which also includes many pest species. The genetic toolbox for <it>Tribolium </it>research has expanded in the past years but spatio-temporally controlled misexpression of genes has not been possible so far.</p> <p>Results</p> <p>Here we report the establishment of the GAL4/UAS binary expression system in <it>Tribolium castaneum</it>. Both GAL4Δ and GAL4VP16 driven by the endogenous heat shock inducible promoter of the <it>Tribolium hsp68 </it>gene are efficient in activating reporter gene expression under the control of the Upstream Activating Sequence (UAS). UAS driven ubiquitous tGFP fluorescence was observed in embryos within four hours after activation while <it>in-situ </it>hybridization against tGFP revealed expression already after two hours. The response is quick in relation to the duration of embryonic development in <it>Tribolium </it>- 72 hours with segmentation being completed after 24 hours - which makes the study of early embryonic processes possible using this system. By comparing the efficiency of constructs based on <it>Tribolium, Drosophila</it>, and artificial core promoters, respectively, we find that the use of endogenous core promoters is essential for high-level expression of transgenic constructs.</p> <p>Conclusions</p> <p>With the established GAL4/UAS binary expression system, ectopic misexpression approaches are now feasible in <it>Tribolium</it>. Our results support the contention that high-level transgene expression usually requires endogenous regulatory sequences, including endogenous core promoters in <it>Tribolium </it>and probably also other model systems.</p

ArgR-Independent Induction and ArgR-Dependent Superinduction of the astCADBE Operon in Escherichia coli

For Escherichia coli, growth in the absence of ammonia is termed nitrogen limited and results in the induction of genes that assimilate other nitrogen sources, a response mediated by σ(54) and nitrogen regulator I (NR(I), also called NtrC). The astCADBE operon, which is required for growth with arginine as the sole nitrogen source, is moderately expressed during general nitrogen limitation and maximally expressed in the presence of arginine. The operon is also induced in stationary phase. Primer extension analysis of E. coli revealed the presence of a σ(54)-dependent promoter utilized in exponential phase during nitrogen limitation and a σ(S)-dependent promoter active during stationary phase. We used an ast-lacZ fusion to show that arginine stimulates expression, that ArgR, the arginine repressor, enhances expression from both promoters but is not essential, and that transcription by the two forms of the RNA polymerase is competitive and mutually exclusive. We demonstrated the binding of RNA polymerase holoenzymes, NR(I), and ArgR to the promoter region in vitro. We also reconstituted transcription from both promoters with purified components, which confirmed the accessory role of ArgR for the σ(54)-dependent promoter. Thus, the ast operon exhibits nitrogen source-specific induction that is unique for an NR(I)-dependent gene. The transcriptional regulation of the ast operon in E. coli differs from that in Salmonella enterica serovar Typhimurium, in which ArgR is required for ast operon expression