7] Ras signaling to transcription activation: Analysis with gal4 fusion proteins

Abstract

This chapter describes the mechanism of Ras signaling for the activation of transcription process. Activation of Ras signaling stimulates multiple downstream signaling cascades, many of which converge on the nucleus and alter cellular gene expression. These widespread changes in gene expression are important for regulating normal growth and, when aberrantly regulated, for mediating oncogenests. Many of the changes in gene expression mediated by Ras are the result of signaling through multiple parallel pathways resulting in activation of mitogen activated protein (MAP) kinase family members. One approach is to transiently contransfect oncogenic Ras and a reporter construct consisting of the promoter region of the gene fused to an easily assayed reporter coding sequence (e.g., luciferase). The type of reporter gene analysis described in this chapter utilizes fusion proteins between a heterologous DNA-binding domain from yeast GAL4 and the trans-activation domain of a specific transcription factor. Overall, this type of reporter gene analysis involves transient contransfection of two or three different plasmids into an appropriate cell line.