Chimeric Transcription Factor Assay

Introduction

In this assay, we investigated whether human cells (EA.hy926 cell) receive AHL, a signaling molecule that is synthesized in and exported from E. coli and induce the transcription of atIPT4 and log1 genes to synthesize iP.

Note

AHLs, which stand for [N-]acyl-homoserine lactones, are small signaling molecules and are employed in the bacterial “Quorum Sensing”. Among several kinds of AHLs, 3OC8HSL(C8) was employed in the assay.
Isopentenyl adenine (iP) is a kind of cytokinin and we use it as a signal molecule from human cells to E. coli in the inter-kingdom communication. Cytokinins are the signaling molecules (or Phytohormones) that plants produce and play important roles in cell growth and differentiation. In the case of Arabidopsis thaliana, extracellular iP is received by a transmembrane receptor, AHK4. AHK4 has a histidine kinase activity, and binding of iP to AHK4 triggers auto-phosphorylation of AHK4 and the following histidine-to-aspartate phosphorelay. As a consequence, transcription from target genes is induced and/or repressed so that physiological states of plants are changed. The histidine kinase activity of AHK4 has shown to be activated depending on iP even in E. coli cells (Suzuki et al. 2001, Lukáš Spíchal et al. 2004). This fact encouraged us to use iP as a signaling molecule in our project (See AHK4 Assay page).

Summary

As shown in Fig.1, two kinds of constructs were introduced into EA.hy926 cells by electroporation. The CAG promoter (pCAG) constantly expresses the chimeric protein, relA/NLS/traR. When relA/NLS/traR binds with C8, this complex binds to (tra box)7 sequence (the enhancer sequence; see below) and activates transcription from the CMV minimal promoter (CMV min). As a result, at the atIPT4 and log1 genes are transcribed depending on C8. The atIPT4 and log1 gene products jointly work as IP synthetase.

Fig1. Construction of iP synthetase genes

Results

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Fig2. Result of the qualitative experiment

Discussion

We confirmed that the transcription of atIPT4 and log1 genes are induced by C8 addition and the degree of induction depends on C8 concentration.

Protocol

Reference

Petra Neddermann, Cesare Gargioli, Ester Muraglia, Sania Sambuncini, Fabio Bonelli, Raffaele De Francesco, Riccardo cortese (2003) A novel, inducible, eukaryotic gene expression system based on the quorum-sensing transcription facter TraR. EMBO reports VOL 4: 159-165.