Versatile Assay System Research Articles

Measuring Hox-DNA binding by electrophoretic mobility shift analysis.

Understanding gene regulation by Hox transcription factors requires understanding the forces that underlie DNA binding by these proteins. Electrophoretic mobility shift analysis (EMSA) not only allows measurement of protein affinity and cooperativity but also permits visualization of differently migrating protein-DNA complexes, including complexes with different compositions or complexes with identical compositions yet assembled in different geometries. Furthermore, protein activity can be measured, allowing correction of binding constants for the percentage of protein that is properly folded and capable of binding DNA. Protocols for measuring protein activity and the equilibrium DNA-binding dissociation constant (K d) are provided. This versatile assay system can be adjusted based on specific needs to measure other parameters, including the kinetic association and dissociation constants (k a and k d) and the formation of heterologous protein-protein interactions.

Non-destructive bioluminescence detection system for monitoring defense gene expression in tobacco BY-2 cells

Using tobacco BY-2 cells transformed with a promoter-luciferase gene fusion, we developed a non-destructive and sensitive in vivo assay system for monitoring defense gene expression in higher plant cells. A promoter fragment of a tobacco salicylic acid (SA) inducible pathogenesis-related gene, PR-1a isolated from the genomic DNA of tobacco BY-2 cells, was fused to the luciferase reporter gene and introduced into plant cells by Agrobacterium-mediated transformation. To detect the PR-1a promoter expression as a luciferase activity, transformed cells were mixed with luciferin solution and the bioluminescence levels were monitored in vivo using a conventional luminometer. Because the PR-1a promoter expression levels of the BY-2 cells are relatively high, the induction of the luciferase activities by the treatment with SA was barely detectable under log phase growth conditions. However, we could observe concentration dependent induction of the luciferase activities following SA application under stationary phase. Treatment with benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester (BTH) and methyl-2,6-dichloroisonicotinic acid (INA) also resulted in a drastic increase in luciferase activities of transgenic cells in a dose dependent manner. On the other hand, treatment with an inactive SA analog 4-hydroxybenzoic acid (4-HBA) showed no influence on luciferase activities. The sensitivity of the assay system was higher than the previously reported techniques for the detection of induction by SA or BTH. These results indicate that this rapid, inexpensive and versatile assay system would be useful for the identification and characterization of chemicals capable of inducing defense gene expression in higher plant cells.