Isolation of transcription factors binding auxin response elements using a yeast one-hybrid system

Plant hormones play an important role during higher plant embryogenesis. Auxin is central to the development of vascular tissues, formation of lateral and adventitious roots, control of apical dominance, and tropic responses. Auxin response element (AuxRE), present in the promoters of many auxin-induced genes, can confer auxin responsiveness. Using carrot somatic embryo under specific developmental phase, a cDNA expression library was constructed. Several plasmids were recombined containing the tetramer of AuxRE as a bait. After screening by a yeast one-hy-brid system, one positive clone was confirmed and characterized. Electrophoretic mobility shift assay showed that AxRF1 protein expressed in yeast cell could bind AuxRE in vitro. It suggests that AxRF1 participates in regulation of the expression of auxin responsive gene during carrot somatic embryogenesis.     

Isolation of transcription factors binding auxin response elements using a yeast one-hybrid system

Plant hormones play an important role during higher plant embryogenesis. Auxin is central to the development of vascular tissues, formation of lateral and adventitious roots, control of apical dominance, and tropic responses. Auxin response element (AuxRE), present in the promoters of many auxin-induced genes, can confer auxin responsiveness. Using carrot somatic embryo under specific developmental phase, a cDNA expression library was constructed. Several plasmids were recombined containing the tetramer of AuxRE as a bait. After screening by a yeast one-hybrid system, one positive clone was confirmed and characterized. Electrophoretic mobility shift assay showed that AxRF1 protein expressed in yeast cell could bind AuxRE in vitro. It suggests that AxRF1 participates in regulation of the expression of auxin responsive gene during carrot somatic embryogenesis.     

Motif X (CACACGTGGG) and motif Y (CACACGTATC) of theDcECP31 promoter have different functions in the ABA-response pathway

We have identified two important elements in the DcECP31 promoter, the ABA-responsive element (ABRE)-like motif X (CACACGTGGG), and motif Y (CACACGTATC), which are sufficient for embryo-specific and ABA-inducible promoter activities. Using gain-of-function analysis, we demonstrated that motifs X and Y are necessary and sufficient to confer ABA-responsiveness upon a heterologous, truncated CaMV minimum promoter. Motif-exchange experiments were carried out to determine whether these motifs exerted similar effects on the DcECP31 promoter. The results suggest that motif X functions as an enhancer-like element and that motif Y participates in ABA-responsiveness.     

Dimerization of the hepatitis C virus nonstructural protein 4B depends on the integrity of an aminoterminal basic leucine zipper

The hepatitis C virus (HCV) nonstructural (NS) protein 4B is known for protein–protein interactions with virus and host cell factors. Only little is known about the corresponding protein binding sites and underlying molecular mechanisms. Recently, we have predicted a putative basic leucine zipper (bZIP) motif within the aminoterminal part of NS4B. The aim of this study was to investigate the importance of this NS4B bZIP motif for specific protein–protein interactions. We applied in silico approaches for 3D‐structure modeling of NS4B‐homodimerization via the bZIP motif and identified crucial amino acid positions by multiple sequence analysis. The selected sites were used for site‐directed mutagenesis within the NS4B bZIP motif and subsequent co‐immunoprecipitation of wild‐type and mutant NS4B molecules. Respective interaction energies were calculated for wild‐type and mutant structural models. NS4B‐homodimerization with a gradual alleviation of dimer interaction from wild‐type towards the mutant‐dimers was observed. The putative bZIP motif was confirmed by a co‐immunoprecipitation assay and western blot analysis. NS4B‐NS4B interaction depends on the integrity of the bZIP hydrophobic core and can be abolished due to changes of crucial residues within NS4B. In conclusion, our data indicate NS4B‐homodimerization and that this interaction is facilitated by the aminoterminal part containing a bZIP motif.