Genomic Maintenance & Repair

Monday, October 17, 2005 3:00 PM-5:00 PM Exhibit Hall

(PP357) Ultrasensitive analysis of protein expression in single Deinococcus radiodurans tetrads.

Turner, Emily*,1, Dovichi, Norman1, 1 Department of Chemistry, Seattle, WA, USA

ABSTRACT- Clonal populations of Deinococcus radiodurans display heterogeneous response to radiation. Stochastic cell-to-cell variation in the production of proteins involved in DNA repair, such as RecA, may contribute to observed differences in cell recovery from irradiation. Following DNA damage, RecA expression levels have been shown to increase across the population, suggesting a mechanism for adaptive response. We are developing instrumentation and methodology based on capillary electrophoresis to analyze changes in expressed proteins, including RecA, in individual tetrads of D. radiodurans. Capable of detecting GFP at the single copy level, this technology provides accurate detection of fluorescently-tagged proteins in a single cell. Our group has developed one- and two-dimensional capillary electrophoresis to perform proteomic analysis of single cells. Laser-induced fluorescence detection provides low zeptomole (10-19) limits of detection, permitting the analysis of single cells. In these experiments, a single tetrad is injected into a silica capillary. On-column lysis is performed with heat and detergents, and cellular proteins are labeled with a fluorescent tag. An electric field is applied to the capillary to separate proteins by mass, charge, or hydrophobicity. We have also created a strain of D. radiodurans that expresses a RecA-EGFP fusion protein, under the control of the recA promoter. The effect of DNA damage on the expression level of the fusion protein in individual tetrads is determined by capillary electrophoresis. These experiments provide accurate and rapid determination of protein expression in individual D. radiodurans tetrads. Development of this technology will allow ultrasensitive detection of any fluorescently-tagged protein in single eukaryotic or prokaryotic cells.

Key words: Deinococcus radiodurans, recA, capillary electrophoresis, adaptive response

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2005 RRS