Functional analysis of the regulation mechanism of viral immediate early gene expressions

Description

The electrophoretic mobility shift assay (EMSA) is an established methodology for studying the interaction between a particular protein and a target DNA probe. In conjunction with mutagenesis, EMSA can be used to identify the important binding sequences within a given gene's upstream regulatory region there we described how this technique was successfully used to study the regulation mechanism of viral immediate early gene expression.

Brief protocol

DNA binding assay (EMSA). For the EMSA to test the ability of transcription factor to bind with the putative binding sequence of the viral immediate early gene promoter, the cell or tissue nuclear extracts were separately mixed with EMSA reaction buffer [4% Ficoll, 12 mM HEPES (pH 7.9), 4 mM Tris-HCl (pH 7.9), 0.1 mM EDTA, 5 μg of poly(dI-dC) and 1 mM dithiothreitol] to a total volume of 15 μl and incubated for 10 min at room temperature. A ³²P-labeled (30,000 cpm) double-stranded ie promoter probe was added, and the mixture was incubated at room temperature for 20 min. For the competition experiments, nuclear extracts were preincubated for 10 min with a 10´ or 40´ molar excess of the unlabeled double-stranded competitor oligonucleotides. After preincubation, the labeled probe was added and the mixtures were incubated as described above (room temperature; 20 min). The reaction mixtures were then separated by 6% polyacrylamide gel electrophoresis, after which the gels were dried and exposed to Kodak Biomax MS film. Supershift EMSA was used to confirm that the EMSA complexes were specifically formed between transcription factor and ie promoter probe. For the supershift assays, nuclear extracts prepared as described above were preincubated for 1 h at room temperature in the presence of appropreciate antibody. The labeled probe was then added to each mixture, and the mixtures were incubated as described above. Separation was by 4% PAGE, and this was followed by drying and exposure to Kodak Biomax MS film.

Some of our publications that have used this platform

1. Liu, W.J., Chang, Y.S., Wang, Andrew.H.J., Kou, G.H., and Lo, C.F. (2007). White spot syndrome virus annexes a shrimp STAT to enhance the expression of the immediate-early gene ie1. J. Virol. 81, 1461-1471.
2. Liu, W.J., Chang, Y.S., Wang, H.C., Leu, J.H., Kou, G.H., and Lo, C.F. (2008). Transactivation, dimerization, and DNA-binding activity of white spot syndrome virus immediate early protein IE1. J. Virol. 82, 11362-11373.