Reverse Transcription-PCR Differential Display Analysis of Escherichia coli Global Gene Regulation in Response to Heat Shock

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American Society for Microbiology

RESUMO

A reverse transcription (RT)-PCR technique was developed to analyze global gene regulation in Escherichia coli. A novel combination of primers designed specifically for the start and stop regions of E. coli genes (based on the findings of Fislage et al. [R. Fislage, M. Berceanu, Y. Humboldt, M. Wendt, and H. Oberender, Nucleic Acids Res. 25:1830–1835, 1997]) was used as an alternative to the poly(T) primers often used in eukaryotic RT-PCR. The validity of the technique was demonstrated by applying it to heat shock analysis. Specifically, RT-PCR-amplified total RNA from heat-shocked and non-heat-shocked cells were hybridized with slot blots of the Kohara set (U. Kohara, K. Akiyama, and K. Isono, Cell 50:495–508, 1987; S. Chuang, D. Daniels, and F. Blattner, J. Bacteriol. 175:2026–2036, 1993). The signals obtained for heat-shocked and control cultures of each clone were compared, and differences in intensity were evaluated by calculating induction ratios. Clones that were considered significantly induced were subsequently mapped by the Southern blot technique in order to determine specific gene upregulation. Also, for several genes, Northern blotting and total RNA dot blotting were performed to confirm that the transcript levels in the original RNA samples were different. This technique extended previously described methods for studying global gene regulation in E. coli by incorporating a PCR amplification step in which global, mRNA-specific primers were used. In addition, the method employed here can be easily extended to study E. coli global gene regulation in response to additional environmental stimuli.

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