EMFID: Environmental monitoring by fluorescence induction and detection

• Research unit: EU RFP
• Project number: 97.0125

Key words (English)

Environmental monitoring; DNA damage; transcription

Alternative project number (English)

EU project number: ENV4-CT97-0480

Research programs (English)

EU-programme: 4. Frame Research Programme - 3.1 Environment

Short description (English)

See abstract

Partners and International Organizations (English)

Coordinator: University of Manchester (UK)

Abstract (English)

The research program 'Environmental Monitoring by Fluorescence Induction and Detection (EMFID)' harnesses DNA damage-inducible promoters of the yeast Saccharomyces cerevisiae directing the expression of a fluorescent marker protein (green fluorescent protein) in order to monitor genotoxic substances in the environment. The project partners are improving the instrumentation and are developing the miniaturization of the detection system. Our focus is on the in vivo regulation of the reporter system, and our aim is to identify novel trans-acting factor involved in DNA damage-induced transcriptional regulation. To this end, we performed two screens that identified 19 different candidate genes. Several novel potential regulators have been identified as well as some known transcriptional regulators that have not yet been linked to this biological context of DNA damage-induced transcription. Further analysis established an astounding complexity in the DNA damage-regulated transcription of several target genes involving repression and activation of constitutive as well induced expression levels. These results have been summarized in a manuscript that has been submitted for publication [L. Walsh, J. Schmuckli-Maurer, N. Billinton, W.-D. Heyer, and R. M. Walmsley (2001) DNA damage-induced transcription of RAD54 can be regulated independently of RAD9 and DDC1 checkpoints]. In order to better characterize the complex interactions during the transcriptional response to DNA damage, we have decided to evaluate new color-shifted fluorescent proteins in addition to the green fluorescent protein of the original assay. The rationale being that the transcriptional response of more than one pathway could be monitored in the same cell, if fluorescent proteins with different emission wavelengths (colors) were used as reporters for different pathways. After a review of the alternative fluorescent proteins currently available four genes encoding fluorescent proteins were selected for trial in our reporter system. Analysis of the nucleotide sequence of these gene revealed that the mammalian codon bias they shared would hinder their expression in yeast. To overcome these limitations, we decided to resynthesize each gene using an optimal codon bias for high expression in yeast. The resulting codon-optimized genes are currently being tested in yeast and promise to be highly valuable tools for all applications of fluorescent proteins in yeast.

References in databases (English)

Swiss Database: Euro-DB of the
State Secretariat for Education and Research
Hallwylstrasse 4
CH-3003 Berne, Switzerland
Tel. [phone]
Swiss Project-Number: 97.0125